Neuronal serine-threonine protein kinase

ABSTRACT

The present invention relates to a gene and the coded protein thereof that is involved in the development of sequelae of local ischaemia. The new protein is a serine threonine protein kinase and provides a new therapeutic approach to the prophylaxis and therapy of apoplexy.

[0001] The present invention relates to a serine threonine proteinkinase, nucleic acids that code this kinase and their use in thediagnosis and therapy of neuronal and neoplastic diseases.

[0002] Apoplexy, also known as stroke, cerebral apoplexy and apoplecticshock, is involved in about 15% of all deaths, with men and women beingaffected equally. Symptoms range from disturbances of consciousness tocoma, and often also encompasses spastic hemiplegia, the most disparatesymptoms of central-motor and sensory loss as well as focal orgeneralised convulsions. Every instance of apoplexy involves acirculatory disorder in the localised cerebral region that is associatedwith oxygen deficiency. Two basic mechanisms act as triggers. Firstly,massive bleeding, encephalorrhagia, which is involved in 15% of cases,with this generally occurring in striolenticular arteries followingvascular rupture, as a result of which limited cerebral regions aredestroyed. This mechanism results in a high degree of lethality. Theprimary disease, which came in question, is in particular hypertonia,arteriosclerosis, intracranial aneurysm and, more rarely, consumptivecoagulopathy. A cerebral infarction, encephalomalacia, is involved asthe second mechanism, with this being regarded as the cause in 85% ofcases. A necrosis is generally formed in this connection. Causes forthis include arterial thrombosis, thromboembolism or functionalischaemia associated with open vascular lumens, e.g. following a drop inblood pressure. The cerebral infarction “ischaemic necrosis” is thecause of the apoplexy in about 70-80% of cases. Arteriosclerosis oftenrepresents the underlying causal disease. The rare, slowly developingsymptoms of encephalomalacia are termed “progressive stroke”. Transientsymptoms of neurological loss without the formation of tissue damage(“transient ischaemic attacks”) should be considered as the early signsof a cerebral infarction. A temporary stenotically induced ormicroembolism-induced limited circulatory disturbance is assumed to bethe cause. Diagnosis encompasses not only a general and neurologicalexamination but also cranial computer tomography, cerebrovascularDoppler ultrasound examination, spinal tap, dynamic brain scanning, EEGand nuclear spin resonance tomography.

[0003] The molecular principles of ischaemia and the associated sequelaeare to date virtually unknown. However, it may be assumed that a complexseries of biochemical processes is required until apoplexy occurs.

[0004] The present invention was intended to address the technicalproblem of identifying genes involved in the development of apoplexyfollowing local oxygen deficiency and thus opening up new approaches tothe prophylaxis and treatment of apoplexy. The present invention wasfurther intended to address the technical problem of identifyingproteins involved in the development of apoplexy.

[0005] The said technical problems are solved by a nucleic acid thatcodes for a serine threonine protein kinase, with the nucleic acid beingselected from:

[0006] a) a nucleic acid with one of the sequences according to SEQ IDNOs. 1-4 and a nucleic acid that codes for a protein with a sequenceaccording to one of SEQ ID NOs. 5-8;

[0007] b) a nucleic acid that hybridises with a nucleic acid accordingto a);

[0008] c) a nucleic acid which, taking account of the degeneration ofthe genetic code, would hybridise with a nucleic acid according to a);

[0009] d) derivatives of a nucleic acid according to a)-c) that areobtained by substitution, addition, inversion and/or deletion of one ormore bases; and

[0010] e) a nucleic acid that is complementary to a nucleic acidaccording to a)-d).

[0011] For example, in derivatives of the proteins according to SEQ IDNOs. 5-8, arginine radicals are replaced by lysine radicals, valineradicals by isoleucine radicals or aspartic acid radicals by glutamicacid radicals, with the physicochemical properties of the replaced aminoacid and the amino acids to be replaced being very similar (e.g. spatialfilling, alkalinity, hydrophobicity). However, one or more amino acidsmay also be replaced within their sequence, added or removed, or severalof these measures may be combined with one another. The proteins thatare thus modified with respect to SEQ ID NOs. 5-8 have at least 60%,preferably at least 70% and particularly preferably at least 90%sequence identity with the sequences SEQ ID NOs. 5-8, calculated inaccordance with the algorithm of Altschul et al., J. Mol. Biol., 215,403-410, 1990. The isolated protein and its functional variants can beisolated advantageously from the brain of mammals such as Homo sapiens,Rattus norvegicus or Mus musculus. The term ‘functional variants’ shouldalso be understood to mean homologues from other mammals.

[0012] The nucleic acids according to the invention according to SEQ IDNOs. 1-4 represent nucleic acids as isolated from the mouse (SEQ ID NOs.1 and 2) or humans (SEQ ID NOs. 3 and 4) respectively, with the nucleicacids according to SEQ ID no. 2 and SEQ ID no. 4 being longer splicevariants of SEQ ID no. 1 and SEQ ID no. 3 respectively. Nucleic acidsthat code for a protein that displays at least 60%, preferably at least70% and particularly preferably at least 90% identity to one of theproteins as coded by SEQ ID NOs. 1-4 are also regarded as being inaccordance with the invention.

[0013] “Derivatives” of the aforesaid nucleic acids according to theinvention, e.g. allele variants, differ from the said nucleic acidsaccording to SEQ ID NOs. 1-4 by substitution, addition, inversion and/ordeletion of one or more bases, but with kinase activity beingmaintained. Derivatives, such as homologues or sequentially alliednucleic acid sequences, can be isolated from all mammalian species,including humans, by current methods via hybridisation with one of thenucleic acid sequences according to the invention or fragments thereof.

[0014] The term “functional equivalents” should also be understood tomean homologues of the nucleic acid according to SEQ ID NOs. 1-4, forexample homologues from other mammals, shortened sequences,single-strand DNA or RNA or PNA of the coding and non-coding nucleicacid sequence. Functional equivalents of this kind can be isolated onthe basis of the nucleic acids of SEQ ID NOs. 1-4, for example bystandard hybridisation methods or PCR technology, from othervertebrates, such as mammals. Oligonucleotides from conserved regionsthat can be determined by the expert in the known way are advantageouslyused for hybridisation. However, longer fragments of the nucleic acidsaccording to the invention or the entire sequence may also be used forhybridisation. Standard conditions for hybridisation vary according tothe nucleic acid used—oligonucleotide, longer fragment or fullsequence—or according to which nucleic acid type—DNA or RNA—is used forhybridisation. Thus, for example, melting temperatures for DNA:DNAhybrids are around 10° lower than those of DNA-RNA hybrids of the samelength.

[0015] The term “standard conditions” should, for example depending onnucleic acid temperatures, be understood to mean between 42 and 58° C.in an aqueous buffer solution with a concentration of between 0.1-5×SSC(1×SSC=0.15 M NaCl, 15 mM sodium citrate, pH 7.2) or additionally in thepresence of 50% formamide, such as for example 42° C. in 5×SSC, 50%formamide. Advantageously, hybridisation conditions for DNA:DNA hybridsare 0.1×SSC and temperatures between around 20° C.-45° C., preferablybetween around 30° C.-45° C. For DNA:RNA hybrids, hybridisationconditions are advantageously 0.1×SSC and temperatures between around30° C.-55° C., preferably between around 45° C.-55° C. These specifiedtemperatures for hybridisation are for example calculated meltingtemperature values for a nucleic acid with a length of approx. 100nucleotides and a G+C content of 50% in the absence of formamide. Theexperimental conditions for DNA hybridisation are described in relevantgenetics textbooks such as, for example, Sambrook et al., “MolecularCloning”, Cold Spring Harbor Laboratory, 1989, and can be calculated byformulae familiar to the expert, for example depending on the length ofthe nucleic acids, the nature of the hybrids or the G+C content.Additional information on hybridisation can be obtained by the expertfrom the following textbooks: Ausubel et al. (eds), 1985, CurrentProtocols in Molecular Biology, John Wiley & Sons, New York; Hames andHiggins (eds), 1985, Nucleic Acids Hybridization: A Practical Approach,IRL Press at Oxford University Press, Oxford; Brown (ed), 1991,Essential Molecular Biology: A Practical Approach, IRL Press at OxfordUniversity Press, Oxford.

[0016] The term “derivatives” of the sequences according to theinvention according to SEQ ID NOs. 1-4 should also be understood to meanpromoter variants upstream of the coding regions of the sequencesaccording to the invention; these may be modified by one or morenucleotide replacements via insertion, addition and/or deletion withoutthe promoter properties, particularly promoter strength andinducibility, being impaired. However, the term “derivative” also coverspromoter variants which, based on the sequences according to theinvention, are strengthened in their activity by nucleotidereplacement(s).

[0017] “Neoplastic diseases” are those to do with abnormal growthbehaviour of cells, the loss of intercellular inhibiting mechanisms,etc. These include, for example, carcinomas as abnormal proliferationsof endodermal cells, lymphoneoplastic diseases, melanomas, etc.

[0018] A preferred nucleic acid codes for a protein with a sequenceaccording to one of SEQ ID NOs. 5-8 or a protein that displays at least60% identity to one of the said sequences.

[0019] In a further preferred embodiment, the nucleic acid is at least60% identical to the coding sections of one of the sequences accordingto one of SEQ ID NOs. 1-4.

[0020] In a further preferred embodiment, the nucleic acid codes for aprotein sequence according to one of SEQ ID NOs. 5-8, with a nucleicacid that codes for SEQ ID no. 7 being particularly preferred.

[0021] The nucleic acid according to the invention is preferably a DNA;however, RNA or PNA are also considered.

[0022] As fragments of a nucleic acid according to the invention, thosesuitable for inhibiting the expression of a serine threonine proteinkinase in the antisense orientation to a promoter followingincorporation in a host cell are preferred in particular. Such fragmentsare preferably at least 10 nucleotides, preferably at least 50nucleotides, particularly preferably at least 200 nucleotides long.

[0023] Constructs according to the invention contain the nucleic acidsequence according to the invention or a fragment thereof in combinationwith other sequences, with which they are usually not associated in thegenome of a host cell. Such “foreign sequences” are preferably geneticcontrol elements, transcription and translation signals (also termed“expression-controlling elements” or sequences derived from vectors).The sequences according to the invention are functionally associatedwith these elements.

[0024] This association may, depending on the desired application, leadto an increase or reduction in gene expression. Host organisms can betransformed with the recombinant nucleic acid constructs thus produced.In addition to these control sequences, the natural control of thesesequences of the actual structure genes may still be present and, whereappropriate, have been genetically modified so that natural control hasbeen eliminated and expression of the genes increased. The geneconstruct may, however, also be constructed more simply, i.e. noadditional control signals are inserted upstream of the sequences andthe natural promoter with its control is not removed. The naturalcontrol sequences may instead be mutated in such a way that no furthercontrol takes place and gene expression is increased. Additionaladvantageous control elements may also be inserted at the 3′ terminal ofthe nucleic acid sequences according to the invention. The nucleic acidsequences according to SEQ ID NOs. 1-4 and/or sequences according to SEQID NOs. 5-8 that code for the corresponding proteins may be present inone or more copies in the gene construct, or be located on separate geneconstructs. Advantageous control sequences are present for example inpromoters such as cos, tac, trp, tet, trp-tet, lpp, lac, lpp-lac, laclq,T7, T5, T3, gal, trc, ara, SP6, I-PR or the I-PL promoter, which arepreferably used in gram-negative bacteria. Other advantageous controlsequences are present for example in the gram-positive promoters such asamy and SPO2, in yeast promoters such as ADC1, MFa, AC, P-60, CYC1 andGAPDH or in mammalian promoters such as CaM kinase II, CMV, Nestin, L7,BDNF, NF, MBP, NSE, beta-globin, GFAP, GAP43, tyrosine hydroxylase,kainate receptor subunit 1 and glutamate receptor subunit B. Inprinciple, all natural promoters with their control sequences asreferred to above may be used. In addition, synthetic promoters can beused advantageously. These control sequences are intended to allow fortargeted expression of the nucleic acid sequences and proteinexpression. This may, for example, depending on the host organism, meanthat the gene is expressed or overexpressed only after induction, orthat it is expressed and/or overexpressed immediately. The controlsequences or factors may in this connection preferably positivelyinfluence and thereby increase expression. Strengthening of the controlelements may thus advantageously take place at transcription level bystrong transcription signals being used as promoters and/or “enhancers”.In addition, however, strengthening of translation is also possible by,for example, stability of the mRNA being improved. The term “enhancers”should be understood to mean for example DNA sequences that bring aboutincreased expression via improved interaction between RNA polymerase andDNA. Other control sequences that can be cited include, for example, the“locus control regions”, “silencers” or any sub-sequences thereof. Thesesequences may be advantageously used for tissue-specific expression. Apreferred embodiment is the combination of the nucleic acid sequenceaccording to the invention with a promoter, with the promoter 5′ beinglocated “upstream”. Other control signals such as 3′-positionedterminators or polyadenylisation signals or enhancers may befunctionally used in the nucleic acid construct. The term should also beunderstood to mean complete vector constructs. These vector constructsor vectors are used for expression purposes in a suitable host organism.The nucleic acids according to the invention and/or the genes for theSer/Thr protein kinase are advantageously inserted in a host-specificvector that allows for optimal expression of the genes in the chosenhost. Vectors are well known to the expert and can for example begleaned from the book Cloning Vectors (Eds. Pouwels P. H. et al.Elsevier, Amsterdam-New York-Oxford, 1985, ISBN 0 444 904018). The term‘vectors’ should, besides plasmids, also be understood to mean all othervectors known to the expert such as phages, viruses such as SV40, CMV,Baculovirus, adenovirus, Sindbis virus, transposons, IS elements,phasmids, phagemids, cosmids, linear or circular DNA. These vectors maybe replicated autonomically in the host organism or chromosomallyreplicated. For integration in mammals, linear DNA is advantageouslyused. Expression of the nucleic acids sequences according to theinvention or of the recombinant nucleic acid construct may beadvantageously increased by increasing the number of gene copies and/orby strengthening control factors that positively influence geneexpression. Thus, a strengthening of control elements may preferablytake place at transcription level through the use of strongertranscription signals such as promoters and enhancers. In addition,however, strengthening of translation is possible by, for example,improving the stability of mRNA or increasing the scanning efficiency ofthis mRNA against ribosomes. To increase the number of gene copies, thenucleic acid sequences or homologous genes may be incorporated forexample in a nucleic acid fragment or in a vector that preferablycontains the control gene sequences assigned to the respective genes orpromoter activity with an analogous effect. In particular, controlsequences that strengthen gene expression are used. The nucleic acidsequences according to the invention may be cloned together with thesequences that code for interacting proteins in an individual vector andthen expressed in the desired organism. Alternatively, each of thepotentially interacting nucleic acid sequences and the sequences thatcode for m30 may also be placed in an individual vector and incorporatedseparately in the organism in question via customary methods such astransformation, transfection, transduction, electroporation or particleguns. In addition, the nucleic acid construct according to the inventionor the nucleic acids according to the invention may also be expressed inthe form of therapeutically or diagnostically suitable fragments. Togenerate the recombinant protein, use may be made of vector systems oroligonucleotides that extend the nucleic acids or the nucleic acidconstruct by specific nucleotide sequences and thus code for modifiedpolypeptides that serve the purpose of simpler purification.Hexa-histidine anchors or epitopes that may be recognised as antigens ofvarious antibodies, for example, are known as “tags” of this kind in theliterature (Studier et al., Meth. Enzymol., 185, 1990: 60-89 und Ausubelet al. (eds.) 1998, Current Protocols in Molecular Biology, John Wiley &Sons, New York).

[0025] All cells that permit expression of the nucleic acids accordingto the invention, their allele variants, their functional equivalents orderivatives or the recombinant nucleic acid construct are in principlesuitable as host cells. The term ‘host cells’ should be understood tomean, for example, bacteria, moulds, yeasts, plant or animal cells.Preferred host cells/organisms are bacteria such as Escherichia coli,Streptomyces, Bacillus or Pseudomonas, eukaryotic micro-organisms suchas Saccharomyces cerevisiae, Aspergillus, higher eukaryotic cells fromhumans or animals, for example COS, Hela, HEK293, Sf9 or CHO cells. Thecombination from the host organism and the vectors appropriate to theorganisms such as plasmids, viruses or phages such as for exampleplasmids with the RNA polymerase/promoter system, the phages I, Mu orother temperate phages or transposons and/or other advantageous controlsequences form an expression system. The term ‘expression systems’should preferably be understood to mean for example the combination ofmammalian cells such as CHO cells and vectors such as pcDNA3neo vectoror HEK293 cells and CMV vector that are suitable for mammalian cells.Cell-free, in vitro expression systems are, however, also considered.

[0026] Mammalian tissue, mammalian organs or transgenic mammals are alsoconsidered as hosts according to the invention for expression of thenucleic acids according to the invention. The said hosts preferablydiffer from the wild type in that, compared with the wild type, theycontain a modified quantity of the protein according to the invention orelse a new protein variant of the protein kinase according to theinvention. However, host organisms in which the naturally occurringnucleic acid that codes for a protein according to the invention hasbeen either completely or partially removed or modified in such way thatit is transcription-inactive are also covered.

[0027] The said organisms preferably contain the nucleic acid accordingto the invention or the fragment according to the invention or theconstruct according to the invention integrated in a position in thegenome that does not match its natural position as found in the wildtype.

[0028] Mice, rats, sheep, cattle or pigs are preferably considered astransgenic mammals, although non-mammalian organisms such as plants arealso considered as recipients of the sequences according to theinvention. Transgenic organisms may also be what are known as knock-outanimals. Transgenic animals may in this connection contain a functionalor non-functional nucleic acid sequence according to the invention or afunctional or non-functional nucleic acid construct. A furtherarrangement according to the invention for the transgenic animalsdescribed above is transgenic animals in whose germ cells or all or partof the somatic cells or in whose germ cells and all or part of thesomatic cells the nucleotide sequence according to the invention hasbeen modified by genetic engineering methods or interrupted by theintroduction of DNA elements. Another possibility for use of thenucleotide sequence or parts of it is the production of transgenic orknock-out or conditional or region-specific knock-out animals orspecific mutations in genetically modified animals (Ausubel et al.(eds.) 1998, Current Protocols in Molecular Biology, John Wiley & Sons,New York and Torres et al., (eds.) 1997, Laboratory protocols forconditional gene targeting, Oxford University Press, Oxford). Viatransgenic overexpression or genetic mutation (zero mutation or specificdeletions, insertions or modifications) by homologous recombination inembryonic stem cells, animal models can be produced that can supplyadditional information on the pathogenesis of apoplexy. Animal modelsthus produced may represent essential test systems for evaluating noveltherapeutic agents.

[0029] The proteins according to the invention can be obtained byexpression of one of the nucleic acids according to the invention in asuitable expression system, with expression systems comprising intactcells preferably being considered. The protein according to theinvention is preferably a protein selected from a protein with one ofthe sequences according to SEQ ID NOs. 5-8 or a protein that is at least60%, preferably at least 70%, particularly preferably at least 90%identical to the said sequences, with the “% identity” with thesequences according to SEQ ID NOs. 5-8 being calculated in accordancewith the algorithm of Altschul et al., J. Mol. Biol, 250, 403-410, 1990.The protein according to the invention and functional variants thereofmay, however, also be isolated from the brain of mammals such as Homosapiens, Rattus norvegicus or Mus musculus. Proteins according to theinvention are also those that can be derived from a protein according toone of SEQ ID NOs. 5-8 by amino acid exchange, with protein kinaseactivity remaining essentially unchanged. For example, amino acids inthe starting protein according to SEQ ID NOs. 5-8 may be replaced bythose with similar physicochemical properties (spatial filling,alkalinity, hydrophobicity, etc.). For example, arginine radicals may bereplaced by lysine radicals, valine radicals by isoleucine radicals oraspartic acid radicals by glutamic acid radicals. However, one or moreamino acids may also be transposed in their sequence, added or removed,or several of the said mechanisms may be combined with one another. Themeasures for modifying a specified amino acid sequence to a desiredsequence are familiar to the expert.

[0030] The production of the antibodies according to the invention thatreact with a protein according to the invention is familiar to theexpert. For this purpose, he may for example fall back on the productionof polyclonal antisera or even hybridoma technology for the productionof monoclonal antibodies.

[0031] Inhibitors according to the invention are low-molecular or evenprotein-like substances that can selectively inhibit or completelyeliminate the protein kinase activity of the protein according to theinvention. The identification and production of suitable inhibitors isquite possible for the expert via the use of conventional protein kinaseassays for screening substances. Suitable screening methods and proteinkinase assays are described below. Suitable substances with desiredbinding affinity can also be identified through the use ofcomputer-assisted drug development (CAD) (cf. for example Böhm, Klebe,Kubinyi, 1996, Wirkstoffdesign, Spektrum-Verlag, Heidelberg). Theinhibitors of protein kinase according to the invention thus identifiedare for example suitable for the prophylaxis and/or therapy of strokeand other neurological (particularly neurodegenerative) or neoplasticdiseases. In the case of the said low-molecular substances, peptides andproteins that can enter into a specific interaction with the proteinkinase inhibitor according to the invention are also considered asinhibitors. Such peptide or protein inhibitors can be identified forexample with the aid of the two-hybrid system or even other assays.These assays permit the delimitation of amino acids that are responsiblefor a specific interaction with other interaction partners. Furthermore,the protein according to the invention and its protein kinase activitycan be simply tested in a test system in which the activity of theprotein is measured in the presence of the substance to be tested.Simple measurement methods (colorimetric, luminometric,fluorescence-based or radioactive techniques) that permit rapidmeasurement of a multitude of test substances are preferably involved(cf. Böhm, Klebe, Kubinyi, 1996, Wirkstoffdesign, Spektrum-Verlag,Heidelberg). The test systems described allow the searching of chemicallibraries for substances that have inhibitory or even activating effectson proteins according to the invention. The signal transduction chainthat is induced in ischaemia and proceeds via the protein according tothe invention can be inhibited with these inhibitors. This allows forthe inhibition or prevention of ischaemic sequelae.

[0032] The intracellular physiological interaction partners of theprotein kinase according to the invention, such as phosphorylationsubstrate, and kinase activity-controlling intracellular interactionpartners can be identified via the two-hybrid selection system mentionedabove.

[0033] The protein quantity and also the activity (e.g. specificphosphorylations) of the proteins with the sequences of SEQ ID NOs. 5-8can be determined with the aid of antibodies. A further object of theinvention is therefore a method for quantifying the protein activity ofa protein with one of the sequences SEQ ID NOs. 5-8. Based on the aminoacid sequences according to SEQ ID NOs. 5-8, synthetic peptides can begenerated that are used as antigens for the production of antibodies. Itis also possible to use the polypeptide or fragments thereof for thegeneration of antibodies. The term “antibodies” should be understood tomean polyclonal, monoclonal, human or humanised or recombinantantibodies or fragments thereof, single chain antibodies or evensynthetic antibodies. The term “antibodies according to the invention orfragments thereof” should in principle be understood to mean allimmunoglobulin classes such as IgM, IgG, IgD, IgE, IgA or theirsubclasses such as the subclasses of IgG or their mixtures. IgG and itssubclasses such as for example IgG1, IgG2, IgG2a, IgG2b, IgG3 or IgGMare preferred. The IgG subtypes IgG1/k or IgG2b/k are particularlypreferred. All shortened or modified antibody fragments with one or twobinding sites complementary to the antigen, such as antibody parts witha binding site formed from light and heavy chain corresponding to theantibodies such as Fv, Fab or F(ab′)2 fragments or single-strandfragments, may be cited as fragments. Shortened double-strand fragmentssuch as Fv, Fab or F(ab′)2 are preferred. These fragments may forexample be obtained enzymatically by cleaving the Fc part of theantibodies with enzymes such as papain or pepsin, by chemical oxidationor by genetic manipulation of the antibody genes. Geneticallymanipulated unshortened fragments may also be advantageously used. Theantibodies or fragments may be used alone or in mixtures. The antibodygenes for the genetic manipulations can be isolated in the mannerfamiliar to the expert, for example from hybridoma cells (Harlow, E. andLane, D. 1988, Antibodies: A Laboratory Manual, Cold Spring HarborPress, N.Y.; Ausubel et al., (eds), 1998, Current Protocols in MolecularBiology, John Wiley & Sons, New York). To this end, antibody-producingcells are drawn in and the mRNA isolated from the cells in the knownway, with adequate optical density of the cells, via cell lysis withguanidine thiocyanate, acidification with sodium acetate, extractionwith phenol, chloroform/isoamyl alcohol, precipitation with isopropanoland washing with ethanol. cDNA from the mRNA is then synthesised withthe aid of reverse transcriptase. The synthesised cDNA may be inserteddirectly or following genetic manipulation, for example by “sitedirected mutagenesis”, introduction of insertions, inversions, deletionsor base exchanges into suitable animal, fungal, bacterial or viralvectors and expressed into the corresponding host organisms. Bacterialor yeast vectors such as pBR322, pUC18/19, pACYC184, lambda or yeast muvectors are preferred for cloning of the genes and expression inbacteria such as E. coli or in yeast such as Saccharomyces cerevisiae.Specific antibodies to the proteins according to the invention maybesuitable both as diagnostic reagents and as therapeutic agents fordiseases in which the protein according to the invention is ofpathophysiological importance.

[0034] The diagnostic kit according to the invention contains one of thenucleic acids according to the invention, a fragment thereof or aconstruct containing this, a protein according to the invention and/oran antibody specific to the protein according to the invention, and alsothe other reagents that usually form part of diagnostic kits. Theseinclude suitable buffer solutions and other detection reagents.

[0035] With the method according to the invention for the diagnosis of arisk of apoplexy or assessment of the course of a cerebral infarction,the patient sample is brought into contact with a nucleic acid accordingto the invention and the nucleic acid hybridising therewith in thepatient sample is determined. An elevated level of nucleic acid thathybridises with the nucleic acid according to the invention is anindicator of an increased risk of occurrence of apoplexy. As analternative to the detection of nucleic acid in the patient sample, thequantity of protein according to the invention can also be determined asan indicator of a risk of apoplexy. The antibodies according to theinvention, for example, may be used for protein detection. An elevatedprotein level in the patient sample investigated is also an indicator ofan increased risk of apoplexy. These assays can also be simply performedquantitatively, with the negative control representing material from ahealthy patient.

[0036] Furthermore, the nucleic acids according to the invention andprotein coded therefrom or oligonucleotides and peptides thereof andantibodies targeted at them may be used for the diagnosis of otherdiseases, particularly neurological or cardiovascular or immunologicalor tumour diseases. These materials may further be used for thediagnosis of genetic predispositions to specific neurological,neoplastic, cardiovascular and immunological diseases. In addition,monitoring of treatment of the said diseases can be performed with thesematerials.

[0037] A method for the qualitative and quantitative detection of anucleic acid according to the invention in a biological sample comprisesthe following steps: a) incubation of a biological sample with a knownquantity of nucleic acid according to the invention or a known quantityof oligonucleotides that are suitable as primer for amplification of thenucleic acid according to the invention, b) detection of the nucleicacid according to the invention by specific hybridisation or PCRamplification, c) comparison of the quantity of hybridising nucleic acidor of nucleic acid obtained by PCR amplification with a quantitativestandard. A method for the qualitative and quantitative detection of aprotein heteromer according to the invention or a protein according tothe invention in a biological sample comprises the following steps: a)incubation of a biological sample with an antibody specifically targetedat the protein heteromer or at the protein according to the invention,b) detection of the antibody/antigen complex, c) comparison of thequantities of the antibody/antigen complex with a quantitative standard.A biological sample from a healthy organism is usually removed asstandard. In particular, the property, as set out below, of nucleic acidaccording to SEQ ID NOs. 1-4 being up-regulated in accordance withspecific pathophysiological stimuli, such as for example cerebralischaemias, can be used here. This concerns for example assessment ofthe course of diseases (such as stroke), assessment of therapeuticsuccess, and graduation of the severity of a disease.

[0038] The pharmaceutical composition according to the inventioncontains a nucleic acid according to the invention, a fragment thereof,a construct containing this, a host cell containing the said items, aprotein according to the invention, an antibody targeted at it and/or aninhibitor according to the invention, where appropriate together withthe customary excipients and carriers.

[0039] Therapeutic applications of the items according to the inventionconcern the modulation of processes connected with the phosphorylationof endogenous proteins. These include the following physiological orpathophysiological processes: influencing of immunological activationprocesses (e.g. activation of monocytes, T cells); influencing of celldeath processes, e.g. cascades that lead to cell death, or of processesthat lead to uninhibited growth. The treatable cell types include inparticular neural cells, tumour cells and cells of the immune system.Lastly, cell interactions in which protein kinases are involved, e.g.cell division, cell differentiation, plasticity and regeneration, can beinfluenced with the materials according to the invention.

[0040] The nucleic acid according to the invention or fragments thereofand the construct containing this, the corresponding host cells and theprotein according to the invention, the antibody according to theinvention and/or the inhibitor according to the invention may inparticular be used for the prophylaxis and/or therapy of neurological,particularly neurodegenerative diseases. These include in particularstroke, multiple sclerosis, Parkinson's disease, amyotrophic lateralsclerosis, heterodegenerative ataxias, Huntington's disease,neuropathies and epilepsies.

[0041] Tumour diseases may also be preferentially diagnosed/treatedtherewith. Examples of such tumour diseases include carcinomas of thecolon, rhabdomyosarcoma and bronchial carcinomas.

[0042] Immunological diseases, including autoimmune diseases, atopiesand/or HIV infections and coinfections, may also be diagnosed/treated byother immunotropic viruses and/or acute and/or chronic lymphaticleukaemia, and/or acute and/or chronic myeloid leukaemia and/or primarychronic polyarthritis and/or Crohn's disease and/or Colitis ulcerosawith the protein kinase according to the invention.

[0043] The nucleic acids according to the invention can also be used aspart of gene therapy in mammals and in particular in humans. In genetherapy, the sequences according to the invention can be introducedeither into the body or parts thereof or the expression of endogenoussubstances can be regulated, as for example by means of antisensetechnology. Oligonucleotides, e.g. with antisense orientation or hybridRNA-DNA oligonucleotides that contain fragments of the sequencesaccording to the invention may be used for this purpose. In addition,viral constructs containing a sequence according to the invention may beused. Lastly, bare DNA containing a nucleic acid according to theinvention or parts thereof may be used.

[0044] The SNPs identified in the nucleic acid according to theinvention are also useful for diagnosis in research into humanhereditary diseases. The nucleic acids according to the invention may beused to isolate and characterise additional genes for homologous mRNAsin the murine and human genome with current methods by homologyscreening and correlate them with already known markers for humanhereditary diseases. This permits the identification of additional genesas the cause of specific hereditary diseases.

DESCRIPTION OF FIGURES

[0045]FIG. 1 shows the principle of the Restriction-MediatedDifferential Display (RMDD) method.

[0046]FIG. 2 Tissue distribution of the protein according to theinvention in the rat: The upper illustration shows a Northern Blotobtained with a mouse probe from the 3′ region of SEQ ID no. 1. Thelower illustration shows the ethidium bromide staining of the RNA gelshown in the upper part. A single band of the sample is detected. By farthe greatest expression of the protein according to the invention isevident in the brain; clear transcripts are also still present in testisand lungs, while the other tissues display only very weak bands.

[0047]FIG. 3 shows the tissue distribution of the protein according tothe invention in humans.

[0048] This shows quantitative PCR with the aid of the LightCyclersystem and use of MTC (multiple tissue cDNA) kits from the companyClontech. The strongest expression is located in the brain and lungs andthe intestinal organs.

[0049]FIG. 4 shows the induction of the protein according to theinvention by focal cerebral ischaemia.

[0050] This shows quantification of 9B5 mRNA with the aid of theLightCycler system following induction of focal cerebral ischaemia inmice. The nucleic acid according to the invention is up-regulated fortwo hours after an ischaemic episode. Six hours after ischaemia, thisinduction is no longer detectable.

[0051]FIG. 5 shows the induction of the protein according to theinvention by focal cerebral ischaemia (in situ hybridisation of murinebrain)

[0052] This shows in situ hybridisation of 9B5 following induction offocal cerebral ischaemia in mice. The nucleic acid according to theinvention is up-regulated for two hours after an ischaemic episode(left=ischaemic=induced side). It is also evident that 9B5 is expressedin neurons. Specific induction is to be found in the cortex and in thehippocampal region.

[0053]FIG. 6 shows the catalytic region of SEQ ID no. 7, where thesymbols have the following meaning: I=phosphate anchor, VIb=catalyticregion, VII-VIII=activation loop, V-XI=catalytic domain with subdomains(after Hanks und Hunter, 1995, FASEB J., 9, 576-596); *=activation site,A=ATP binding site; C=active centre.

[0054]FIG. 7 shows the physiological tree for homologous proteinkinases. H9B5 is the phylogenetically youngest protein and the mostclosely related to P78/c-TAK 1. The early cleaving of the C. elegansprotein Par-1 is also clearly shown.

[0055]FIG. 8 shows the domain structure of 9B5.

[0056]FIG. 9 is a hydrophobicity blot according to Kyte-Doolittle.

[0057]FIG. 10 shows the genomic structure of 9B5 in humans.

[0058] This shows the 18 exons of the 9B5 gene. Exon 16 is subject todifferential splicing. The variant 9B5 is formed without exon 16; thevariant 9B5A contains exon 16.

[0059] Protein Sequence of 9B5

[0060] The murine protein sequences (SEQ ID NOs. 5 and 6) are incompleteat the 5′ terminal. In the case of the human sequences according to SEQID NOs. 7 and 8, a full-length clone is involved. This also arises fromalignments with homologous protein kinases.

[0061] In the case of h9B5 (SEQ ID no. 7), an open reading frame resultsthat codes for a protein with 752 amino acids (82.5 kD molecular weight;isoelectric point at pH 9.7). The program PSORT II yields no significantsignal sequences, and does not predict transmembrane regions. AKyte-Doolittle hydrophobicity plot (FIG. 9) also shows a high proportionof hydrophilic regions and only one relatively large hydrophobic region(approx. aa 240-255) which does not, however, seem sufficientlyhydrophobic for a transmembrane region (index>−3). The reportedmembrane-associated location of a number of related proteins(p78/c-TAK1, par1) is, however, interesting in this context.

[0062] An open reading frame of 688 amino acids with a molecular weightof 75.3 kD and an isoelectric point at pH 9.8 results for the sequenceh9B5_b (SEQ ID no. 8). As a result of the insertion of exon 16, a frameshift takes place that leads to an earlier translation stop.

[0063] Classification of 9B5

[0064] A comparison with known protein kinases permits classification ofthe protein according to the invention to the subgroup “CaMK Group II”according to Hanks(http://www.sdsc.edu/Kinases/pkr/pk_catalytic/pk_hanks_seq_align_long.html),(cf. Hanks and Lindberg, Methods Enzymol, 200, 525-32, (1991); Hanks andHunter, Faseb J, 9, 576-96, (1995))). Drewes et al. (Drewes, et al.,Cell, 89, 297-308, (1997)) have already postulated for MARK1/MARK2 andp78 a new subgroup in the already defined SNF1/AMPK subgroup of theCaMKII group. Based on alignment of the most highly homologous proteins(9B5, Par1, cTAK1/p78, MARK1, EMK) and the phylogenetic tree (FIG. 7),the possibility exists that 9B5 defines a new subgroup. At thecarboxy-terminus, 9B5 deviates sharply from the other protein kinases.The kinases in this group are characterised by a highly conservedcarboxy-terminus domain that terminates with the sequence “ELKL”. EMKhas therefore also been designated as “ELKL motif kinase”. It should beassumed that protein kinases still exist that are similar to 9B5 in thecarboxy-terminus and that can be identified via homology screens. Asearch with the program “tblastn” against the EMBL nucleotide databasehas to date yielded no evidence of already known homologues in thecarboxy-terminus region.

[0065] Proteins with the target sequence KXGS and/or KVGS may form thephysiological substrate of the protein according to the invention.Accordingly, the Tau protein or the proteins MAP1 and 2 may represent aphysiological substrate of the protein kinase according to theinvention.

[0066] Subdomains of the Catalytic Domain: 9B5 is a Serine ThreonineProtein Kinase

[0067] The important catalytic subdomains can be clearly defined viacomparisons with other protein kinases (see FIG. 6). The activationsites can likewise be shown (a threonine and a serine that may bephosphorylated; pos. 211 and 215 in h9B5, FIG. 6). A mutation of theseamino acids, for example to alanine, leads to constitutive inactivation.Mutation of the lysine radical (ATP binding) in subdomain II (see FIG.6) likewise leads to inactivation. This finding can be utilised forfurther experiments, e.g. using dominant negative effects. A mutation ofpos. 211 (Ser) and 215 (Thr) to glutamate or aspartate should lead toconstitutive activation as the negatively charged groups may imitatephosphorylation (Huang and Erikson, Proc Natl Acad Sci USA, 91, 8960-3,(1994)). These mutants may be exploited for overexpression of thespecific kinase activity in cells or transgenic animals.

[0068] Subdomain I is known to act as a kind of clasp that anchors thenon-transferrable phosphate groups of ATP. In this connection, thepeptide sequence GKGNFAKV in 9B5 fits in well with consensus motifGXGXXGXV (Hanks and Hunter, Faseb J, 9, 576-96, (1995)). In subdomainII, chiefly lysine (labelled “A” in FIG. 6) is important; this anchorsand orientates the alpha and beta phosphates of ATP and is essential forenzyme function. Subdomain VIb is characterised by the consensussequence HRDLKXXN. This is also very well preserved in 9B5: HRDLKAEN.

[0069] Aspartate (D) is in this connection probably the proton acceptorfor the attacking hydroxyl group during the phosphotransfer reaction(Hanks and Hunter, Faseb J, 9, 576-96, (1995)). Subdomain VII forms withMg2+ ions a chelate complex that encloses the gamma-phosphate and thusorientates this group for the transfer. The sequence DFG is practicallyinvariable. The preserved sequence APE in subdomain VIII is similarlymaintained. This domain is responsible for peptide recognition.Inhibitor peptides can also bind here. Many protein kinases areactivated by phosphorylation within this subdomain. This domain is fullypreserved in EMK, p78 and MARK1. In the case of MARK1, it has alreadybeen shown by direct sequencing that threonine (T) and serine (S) can bephosphorylated, and MARK1 is thereby activated. Owing to thispreservation, it may be assumed with the utmost probability that 9B5also phosphorylates at these sites and thus can be controlled.Autophosphorylation possibly takes place; activation by another kinaseis also conceivable. The tyrosine (Y) in this domain is possibly also aphosphorylation site (as for example in the case of Erk1/2). SubdomainIX is also involved in peptide binding (hydrophobic interaction).

[0070] On account of this structural development, it can be assumed withcertainty that 9B5 is an active protein kinase (serine threoninekinase).

[0071] Tissue Expression of 9B5

[0072] The tissue expression of 9B5 has been investigated in mice andhumans. In mice, a “multiple tissue northern” was performed with varioustissues (FIG. 2). A fragment from the 3′ region of the mouse cDNA wasused as sample. Here, expression takes place in the brain, testicles andlungs.

[0073] In humans, a “multiple tissue northern” (Clontech) was initiallyperformed with 2 different probes (from the 3′ and 5′ regions). However,no clear signal could be obtained, which was attributed to the lowincidence of 9B5 in humans. A quantitative PCR was therefore performedwith the aid of the LightCycler (Roche Diagnostics, Mannheim). cDNAsamples of 8 human tissues already quantitatively standardised on 4different housekeeping genes were used (manufactured by Clontech).Plasmid h9B5-663, which contained an amplified fragment of human9B5-cDNA, was used as control. A touchdown protocol was chosen as PCRprogram. Amplification of a product with a melting point of 90° C. tookplace in all tissue samples; this coincided with the control PCR. Asubsequent gel analysis of the fragments yielded a product of approx.660 bp, i.e. the expected size. 9B5 in humans is actually not verystrongly expressed; amplification becomes visible in the LightCycleronly at around cycle 32. The following primers were used: seq_h9b5_s1GTTGCCATCAAGATTATC (in exon 3) seq_h9b5_a4 CATGATTTGCTCGAGAGTAC (in exon9)

[0074] Quantitative analysis (FIG. 3) shows a relatively ubiquitoustissue distribution with higher concentrations in the brain and lungs,liver, kidneys and pancreas.

[0075] Regulation by Focal Cerebral Ischaemia, a Stroke Model

[0076] The serine threonine kinase 9B5 according to the invention wasidentified by a method for the cloning of differentially regulated genes(RMDD) in the ischaemic hemisphere of mice following focal cerebralischaemia. The animal model for focal cerebral ischaemia represents avalid model for human ischaemic stroke. To bring about the focalcerebral ischaemia, use was made of the so-called thread model, in whicha coated nylon thread is passed through the A. carotis interna up to theend of the A. cerebri media and induces an ischaemic stroke (Clark etal., Neurol. Res., 19, 641-648, (1997)). In cerebral ischaemia,regulation of gene expression plays a crucial role in the course andextent of neuronal damage (Koistinaho and Hokfelt, Neuroreport, 8,i-viii, (1997, Schneider et al., Nat Med, 5, 554-9, (1999)). Inparticular, “immediate early” genes play a role here (Atkins et al.,Stroke, 27, 1682-1687, (1996)), such as cox-2, (Nogawa et al., J.Neurosci., 17, 2746-2755, (1997)).

[0077] 9B5 expression was, following focal cerebral ischaemia,investigated over three timescales: firstly, in a transient ischaemiafollowing two reperfusion periods (ischaemia for 90 mins, reperfusionfor 2 h and 6 h), and secondly in a permanent ischaemia of 24 h (FIG.4). RNA was extracted from the two hemispherical halves of 3-4 brainswithout brain stem and cerebellum (Fasttrack kit, Invitrogen). With theaid of the LightCycler™ system (Roche Diagnostics, Mannheim), aquantitative PCR was performed. The cDNA content of the samples wasstandardised to the expression of cyclophilin and S20 (Schneider et al.,Proc Natl Acad Sci USA, 92, 4447-51, (1995)). The primers used for theamplification of cyclophilin were: cyc5 ACCCCACCGTGTTCTTCGAC acyc300CATTTGCCATGGACAAGATG

[0078] and for the amplification of murine 9B5:

[0079] 9_B5_(1)_(—)1s TATGATCGAACCTCCTTCATGCC

[0080] 9_B5_(1)_(—)1a ATGTCCAGAACTGGGCCTAGCG (These primers amplify anamplimer of 556 bp, which is located at the 3′ terminal of the murinecDNA).

[0081] In actual fact, clear up-regulation of 9B5-RNA by a factor of 7-8on the ischaemic (left) half of the brain appears 2 h after theischaemic event (middle cerebral artery occlusion for 90 mins andreperfusion for 2 h; (FIG. 4); the error bars show standarddeviations—these arise from measurements with thrice serially dilutedcDNA samples and thus reflect the reliability of the measurementresults). After 24 h (in a permanent model), on the other hand, nodifference was any longer detectable.

[0082] In-situ hybridisation was also performed with a 1.6 kb long probefrom the 3′ region of murine 9B5-cDNA (sense and antisense in eachcase). The procedure adopted for this was essentially as per theprotocol of the company Roche Diagnostics (digoxigenin system) followingmodifications by Rossner et al. (Mol Cell Neurosci, 9, 460-475, (1997)).Clear induction is apparent on the ischaemic side (left), particularlyin neurons of the hippocampal region and cortex, less so in the thalamus(FIG. 5). It also becomes clear that 9B5 is predominantly expressed inneurons, and is subject to control there.

[0083] This shows that 9B5 plays an important role in the pathogenesisof stroke, with the up-regulation of 9B5 playing a similar role to that,for example, of the known serine threonine kinases (e.g. JNK, p38).

[0084] A multitude of serine threonine kinases are involved in celldeath processes (e.g. ASK1 (Tobiume, et al., Biochem Biophys Res Commun,239, 905-10, (1997, Berestetskaya, et al., J Biol Chem, 273, 27816-23,(1998, Chen, et al., Oncogene, 18, 173-80, (1999)), DAP (Inbal, et al.,Nature, 390, 180-4, (1997, Levy-Strumpf and Kimchi, Oncogene, 17,3331-40, (1998)), DRAKs (Sanjo, et al., J Biol Chem, 273, 29066-71,(1998)), ZIP (Kawai, et al., Mol Cell Biol, 18, 1642-51, (1998))), DRP-1(Inbal, et al., Mol Cell Biol, 20, 1044-54, (2000)).

[0085] This up-regulation is evidence of a new transcriptional controlmechanism for protein kinases, to date the only known example of this inthe mammalian system. Interestingly, the up-regulation of several MAPkinases was only very recently found in a systematic study of the yeasttranscriptome (Roberts, et al., Science, 287, 873-80, (2000)). Thismight represent a new general mechanism for the regulation of proteinkinases.

[0086] Pharmacological Significance of 9B5 in Neurodegenerative,Neoplastic and Other Diseases

[0087] Approaches to inhibiting/influencing signal transduction pathways“downstream” of a membrane-based receptor have recently been gaining inimportance in pharmacological research. These approaches will in futureprobably play an important part in the treatment of human diseases,particularly in the case of diseases that have hitherto been poorlytreatable or untreatable (Kletsas and Papavassiliou, Exp. Opin. Invest.Drugs, 8, 737-746, (1999)). Advantages of these approaches are that theycan first influence events that can be brought about by several stimuli,and have a common terminal section; secondly, cellular events temporallyfollow the triggering stimulus, and are thus open to intervention forlonger.

[0088] Examples of successful interventions in such signal cascadesinclude inhibitors for caspases that can block apoptosis processes foreven longer after a triggering stimulus. The transcription factorNF-kappaB and these activating processes have also attracted particularattention. For example, cloning of the I-kappaB kinases was pursued withthe aim of finding specific inhibitors for NF-kappaB-mediated genetranscription. Differential transcription profiling has recently beengaining in importance in pharmaceutical research to identify possiblenew points of departure for drugs. Transcriptional control, i.e. controlof the quantity of mRNA in a gene in the cell, is an essential stage inthe cell's response to stimuli, in addition to protein phosphorylations,protein degradation, etc. 9B5 is evidently subject to very rapid controlby transcriptional activation, as shown above. Rapidly controlled genesof this kind are often involved in critical key positions for cellularprocesses.

[0089] A pharmacological influence on 9B5 is possible in the followingway: 1. an effect on the quantity of transcript in the cell, for examplesuppression of rapid up-regulation following pathological processes(e.g. via antisense technology); 2. inhibition of the enzymatic activityof 9B5, particularly kinase activity (e.g. via a kinase inhibitor; 3.inhibition of an interaction with one or more other molecules, e.g.downstream protein kinases, or adaptors.

[0090] Specific inhibitors for a number of MAP protein kinases have beendeveloped recently. One example is PD98059, an inhibitor of MEK1. Thisprevents the phosphorylation of Tau by stimulation with beta-amyloid.This has possible significance for the treatment of Alzheimer's disease.

[0091] The antitumour substance UCN-01 is an inhibitor of cdc25cphosphorylation (Graves, et al., J Biol Chem, 275, 5600-5, (2000)).

[0092] Inhibitors of the protein kinase according to the invention canbe simply identified with conventional protein kinase assays andrepresent an effective aid in controlling the sequelae of cerebralischaemia.

[0093] The experimental data on 9B5 prove its central involvement inprocesses associated with neuronal cell death, excitation, plasticityand neurogenesis. The protein according to the invention represents animportant target molecule for inhibiting or reducing the sequelae offocal ischaemia. Besides this central role in the development ofapoplexy as a consequence of focal ischaemia, the protein according tothe invention might also be an important target molecule in thetreatment of neoplastic diseases, such as cancer. Here, too, the proteinaccording to the invention or its gene might represent the targetmolecule for anticancer agents. The gene might likewise play animportant role in the diagnosis and therapy of cardiovascular diseasesas a number of shared mechanisms exist for ischaemically induceddiseases.

[0094] In a particularly preferred embodiment according to theinvention, the level or activity of the protein kinase according to theinvention is lowered for the prophylaxis and therapy of stroke. This mayfor example take place at the level of expression, byexpression/translation of the corresponding nucleic acids beinginhibited or reduced, or at the level of protein activity, by proteinkinase activity being reduced or inhibited by suitable inhibitors.

[0095] The nucleic acid according to the invention and the protein codedtherefrom open up new therapeutic approaches. Thus, for example, thelevel of endogenous nucleic acid can be influenced by either directlyinfluencing its transcription or even influencing its translation toprotein according to the invention. For example, gene therapy approachesare considered for this in which, via co-suppression or antisensetechnology, the translation of endogenous transcripts is lowered. Newapproaches also present themselves at the level of protein activity bythe protein according to the invention for example representing thetarget molecule of pharmaceutical active substances. Thus, the activityof protein kinase according to the invention can be modified bypharmaceutical active substances in order to intervene in a controllingway in the mechanism of apoplexy. The protein kinase inhibitorsmentioned above represent a class of such pharmaceutical activesubstances. In principle, provision of the nucleic acid according to theinvention or the protein according to the invention thus also opens upentirely new approaches to the prophylaxis or therapy of stroke.

[0096] The following examples elucidate the invention:

EXAMPLES Example 1 Molecular Cloning of 9B5

[0097] Induction of the Thread Model in Mice

[0098] To induce focal cerebral ischaemia in c57/bl6 mice, 3-month-oldmice were used. Following induction of an inhalation anaesthesia (70%N₂O, 30% O₂, 0.8-1% halothane), a 5-0 prolene thread (manufactured bythe company Ethicon) coated with 0.1% poly-L-lysine was passed via theA. carotis externa into the A. carotis interna up to the end of the A.cerebri media. The correct position of the thread is indicated by a dropin the laser Doppler signal (Perimed company) to 10-20% of the startingsignal. Following the performance of this operation and, whereappropriate, determination of additional physiological parameters (bloodpressure, pulse, blood gases, blood glucose), the mice wake from theanaesthesia. After specific occlusion periods, the mice are againsubjected to anaesthesia, and the thread is withdrawn. Reperfusion ofthe tissue thereby takes place. Following specific reperfusion periods,the mice are sacrificed by breaking their necks, and the brainsimmediately prepared and frozen in dry ice.

[0099] In the present case, no reperfusions were performed, only anocclusion for 90 mins or 24 h.

[0100] Preparation of mRNA from the Brains

[0101] The mRNA preparation kit manufactured by Invitrogen (Fasttrack)was used for this purpose.

[0102] Performance of the RMDD Protocol (see also FIG. 1)

[0103] The procedure adopted was essentially as per Pat. No. EP 0 743367A2; U.S. Pat. No 5,876,932, with the modification that 2 μg polyA-RNAwas used for the first-strand synthesis. Following performance offirst-strand, second-strand synthesis, MboI restriction, ligation withadaptors is performed. Two successive PCR reactions with subsets ofprimer combinations follow. The PCR reactions are then loaded onto adenaturing polyacrylamide gel and blotted on a nylon membrane(manufactured by GATC). The biotin-labelled bands are visualised withthe aid of an ordinary streptavidin peroxidase reaction. PCR samples ofthe ischaemic and contralateral hemisphere were applied together to thegel (24 h MCAO on the right and left and 90 mins MCAO on the right andleft). Bands of differing intensity in the right or left hemisphere arecut out, and reamplification of the corresponding PCR product performed.Amplified products obtained are cloned into TOPO TA vector pcDNA 2.1(manufactured by Invitrogen) and sequenced with T7 and M13rev primers(ABI 3700 capillary electrophoresis sequencer).

Cloning of 9B5

[0104] During the performance of this method, a sequence was noticedthat seemed to be up-regulated after 90 mins on the ischaemic side. Thiswas called 9B5. A LightCycler analysis confirmed rapid regulation afterMCAO (90 mins MCAO and 2 h reperfusion) (FIG. 4).

[0105] The isolated 3′-positioned PCR fragment was used to hybridise amurine brain bank, in which there were several clones that containedsequence parts of SEQ ID no. 1 and SEQ ID no. 2. A mouse sample from the5′ region was used to screen a humane foetal brain bank in lambdaZapII(Stratagene). In this connection, 2 different sequences also resultedfrom several clones, which presumably represent splice variants of thesame gene (SEQ ID NOs. 3 and 4), resulted from several clones.

[0106] The detailed production of the human cDNA library used is set outbelow:

Production of the Human cDNA Library

[0107] Using the cDNA synthesis kit manufactured by the companyStratagene, corresponding cDNA libraries were, on the basis of 2 μghuman foetal brain mRNA (manufactured by Clontech) and 5 μg mRNA,produced from adult murine brain, with the procedure adopted essentiallybeing in accordance with the manufacturer's details. To synthesise thefirst-strand cDNA, an oligodT primer was used in accordance with themanufacturer's details. The cloning-compatible (EcoRI/Xhol)double-stranded cDNA fragments were selected by size (in accordance withmanufacturer's details/Stratagene) and ligated into the plasmid vectorpBluescript SKII (Stratagene). The ligation was transformed byelectroporation in E. coli (DH10B, Gibco) and amplified on LB ampicillinagar plates. The plasmid DNA was isolated via alkaline lysis and ionexchanger chromatography (QIAfilter kit, manufactured by Qiagen).

[0108] The complexity of individual clones was 4 million for the foetalhuman brain cDNA bank. From each cDNA bank, 24 individual clones wererandomly analysed by insert sizes, which showed a size distribution of800 bp to 4.5 kB; the mean length of the cDNA inserts was for the humanbank approx. 1.2 kB.

Example 2 Performance of a Reporter Gene Assay

[0109] The sequence of 9B5 obtained can be used to obtain information onthe arrangement of the protein in signal transduction cascades. To thisend, the open reading frame of the gene is cloned into a currentexpression vector (e.g. pCMV-tag, manufactured by Stratagene). Thisconstruct can be transfected with other constructs together ineukaryotic cells (e.g. by the calcium phosphate method, see Ausubel etal., Current Protocols in Molecular Biology, New York, 1997). These maybe reporter constructs, e.g. a luciferase gene under the control of aminimal promoter with several binding sites for, for example, thetranscription factor NF-kappaB or AP-1. Extracts from the cells may thenbe subjected to measurement in the luminometer (e.g. manufactured by thecompany Bertold). An increase in the luciferase value indicatesinfluencing of the signal transduction pathway that results in theactivation of a specific transcription factor. Combinations withexpression constructs for other genes (e.g. MAP kinases) may provideinformation on the precise position of 9B5 in signal cascades. Thesereporter assays can also be performed with other systems, e.g. lacZ orchloramphenicol transferase (CAT assays), without the principle of theassays being influenced.

[0110] In the same way, ready-made kits (e.g. Mercury in vivo kinaseassay kits, manufactured by Clontech) may also be used, with the Tetrepressor being expressed in fusion with the transactivator domain of aphosphorylation target (transcription factors, e.g. Jun). Activation ofa luciferase construct under the control of a Tet repressor element onlytakes place if specific phosphorylation of the transactivator domain bya kinase (e.g. 9B5) occurs. In this way, arrangement in a cellularsignal transduction pathway is possible.

Example 3 Kinase Assays

[0111] Protein kinases are biochemically very well characterised. Thekinase activity of 9B5 can be demonstrated by cloning the open readingframe of 9B5 into an expression vector with an epitope tag (e.g.pcDNA-myc-his) and transfecting it in eukaryotic cells (e.g. Cos cells).After 48 h, extracts can be obtained from these cells andimmunoprecipitation performed with a myc-specific antibody and proteinAbeads. In a kinase buffer in the presence of γ-³²P-ATP, a kinasereaction is performed. The proteins are then denatured and separated onan SDS-PAGE gel. Autoradiography is then performed. Labelled bandsindicate the kinase activity of 9B5. Autophosphorylation of the kinaseitself often takes place as well. Indications of possiblephosphorylation targets are also provided by cotransfections withpotential targets, e.g. various MAP kinases that are also provided witha tag and can be immunoprecipitated.

[0112] Kinase assays may also be performed with 9B5 that has beentranscribed/translated in vitro (e.g. T7 reticulocyte systemmanufactured by the company Promega). Protein that has been expressed inE. coli, e.g. as GST fusion protein or as HIS-tagged construct, can alsobe used. These tags may in this connection be used for purifying theprotein. The purified proteins can then be incubated in kinase bufferwith potential substrates. MBP (myelin basic protein) that is frequentlynon-specifically phosphorylated by Ser/Thr kinases is often used here.The kinase domain of protein kinases is very well defined. The mutationof individual amino acids in the phosphate transfer domain is oftensufficient for loss of function of the protein (e.g. K709M in the caseof ASK1 (Chang et al., Science, 281, 1860-3, (1998)); K90A in DRAK 1,K62A in DRAK2 (Sanjo et al., J Biol Chem, 273, 29066-71, (1998));KK429-430AA in NIK (Sanjo et al., J Biol Chem, 273, 29066-71, (1998));K63W in TAK1 (Ninomiya-Tsuji et al., Nature, 398, 252-6, (1999))). Theseinactive kinases are often of great importance as dominant-negativeinhibitors for evaluation of the cell pathways, e.g. in cotransfectionexperiments and kinase assays (Ninomiya-Tsuji et al., Nature, 398,252-6, (1999)). Mutation of this kind can also be performed with 9B5.

[0113] The specific phosphorylation of target protein can also bedemonstrated with phosphorylation-specific antibodies, e.g.Phospho-SerThr/Tyr monoclonal antibody, mouse IgG2b, produced by thecompany Clontech.

[0114] Further examples of kinase assays that are commonly used in thetechnical arena can, for instance, be gleaned from the book ProteinPhosphorylation, A practical approach, ed. D. G. Hardie, 2nd ed.,Oxford, 1999, particularly chapters 9 and 10.

Example 4 Identification of the Phosphorylation Target of 9B5

[0115] The identification of phosphorylation targets of 9B5 may, forexample, take place via interaction screening. Somewhat classic peptideexpression banks in the lambda bacteriophages (the most used system isthe vector lambda-gt11, see Ausubel et al., Current protocols inmolecular biology, New York, 1997) may be used in this connection. Oneapproach is the cloning of 9B5 into a bacterial expression vector withthe incorporation of a purification tag (e.g. poly-histidine or GST) anda consensus phosphorylation site for protein kinase A (sequence RRASV).9B5 may thus be expressed and purified in bacteria in accordance withstandard methods. 9B5 can in this way be labelled as a scavenger with³³P or ³²P by means of incubation with protein kinaseA. The expressionbank can then be incubated with the labelled 9B5. Following exposure onautoradiograms, positive clones can be identified and be isolated andsequenced by standard methods. This technique can also be adapted in thefollowing way: the autophosphorylation property of 9B5 can be utilisedto label 9B5 with ³³P-γATP via simple incubation. The method can also bemodified in such a way that the expression bank is incubated withpurified 9B5 and ³³P-γATP under phosphorylation conditions(phosphorylation buffer, etc.), and expressed peptides are activelylabelled by 9B5. However, this method is more unstable than thatdescribed above. Sequenced peptides can be used to formulate a consensusrecognition and phosphorylation sequence. This permits theidentification of potential substrates via bioinformatic methods.Candidates can be verified by expression and incubation with 9B5.Examples of the successful performance of these forms of expressionscreening are contained in (Mochly-Rosen and Gordon, Faseb J, 12, 35-42,(1998, Blanar and Rutter, Science, 256, 1014-8, (1992, Chapline et al.,J Biol Chem, 268, 6858-61, (1993, Chapline et al., J Biol Chem, 271,6417-22, (1996, Kaelin et al., Cell, 70, 351-64, (1992, Songyang et al.,Curr Biol, 4, 973-82, (1994)).

[0116] The phosphorylation target of 9B5 can also be identified in ayeast-two-hybrid screen (Fields and Song, Nature, 340, 245-6, (1989)).For example, the interaction of Ras and c-Raf (a Ser/Thr kinase) wasdiscovered in a y2h system (Fields and Song, Nature, 340, 245-6,(1989)). Interaction of the Ser/Thr kinase SNF1 with SNF4 is alsovirtually a prototype for the y2h system (Fields and Song, Nature, 340,245-6, (1989)). In principle in an equivalent way to the yeast screens,mammalian systems can also be used (Fields and Song, Nature, 340, 245-6,(1989)). In the case of a y2h screen, the open reading frame of 9B5 iscloned into a so-called “bait vector” with the GAL4 binding domain (e.g.pGBT10, manufactured by Clontech). A so-called “prey-library” in a yeaststrain can thus be searched for interacting proteins in accordance withseveral current protocols. It can in this connection often be useful touse kinase-negative mutants as these often interact in a more stablemanner with the phosphorylation target. Serine threonine kinases in asynthesis pathway may be brought into spatial proximity by adaptermolecules in order to be able to perform specific phosphorylationsbetter (Chang et al., Science, 281, 1860-3, (1998)), (Yasuda et al., MolCell Biol, 19, 7245-54, (1999, Whitmarsh and Davis, Trends Biochem Sci,23, 481-5, (1998, Whitmarsh et al., Science, 281, 1671-4, (1998)). It istherefore also possible to encounter the phosphorylation targets via twosteps in the yeast-two-hybrid system by first cloning an adaptor proteinand finding the specific target molecule with this as “bait”. All inall, mapping experiments for interaction domains can also be performedwith the y2h system.

[0117] It is also possible to use co-immunoprecipitations from cellstransfected with 9B5 expression vectors to purify proteins binding tothem, and to identify the genes via protein sequencing methods (e.g.MALDI).

[0118] It is also possible, following immunoprecipitation with asubsequent kinase assay from a cell extract, to purify thephosphorylated bands and to sequence these.

[0119] Further examples of the identification of protein kinasesubstrates that are commonly used in the technical arena can be gleanedfrom, for instance, the book Protein Phosphorylation, A practicalapproach, ed. D. G. Hardie, 2nd ed., Oxford, 1999.

Example 5 Apoptosis Assays

[0120] Very many previously identified serine threonine kinases areinvolved in apoptotic processes, e.g. ASK1 (Zhang et al., Proc Natl AcadSci USA, 96, 8511-5, (1999)), (Ichijo et al., Science, 275, 90-4,(1997)), DRAKs (Zhang et al., Proc Natl Acad Sci USA, 96, 8511-5,(1999)), (Ichijo et al., Science, 275, 90-4, (1997)). The involvement of9B5 in apoptotic cascades can be further investigated by transfectingexpression constructs with 9B5 in eukaryotic cells, and theninvestigating the induction of apoptosis. This may, for example, takeplace via staining with annexin (manufactured by Roche Diagnostics), byantibodies that recognise the active form of caspase-3 (manufactured byNew England Biolabs), or by ELISAs that recognise DNA-histone fragments(cell-death elisa, Roche Diagnostics). This induction of apoptosis maybe cell type-specific, and so several cell lines and primary cells mustbe investigated. The induction of apoptosis may also bestimulus-specific, and so several stress situations may be helpful inanswering this question, e.g. heat shock, hypoxia conditions, cytokinetreatments (e.g. II-1, II-6, TNF-alpha), H₂O₂ treatment. On cell types,several customary lines, e.g. Cos cells, HEK cells, PC12 cells, THP-1cells and primary cells such as, for example, neurons and astrocytes areconsidered, as are other immortalised and primary cell lines, asrequired.

Example 6 High-Throughput Screening Assays for the Identification ofInhibitors of 9B5

[0121] 9B5 can be used to find inhibitors of the interaction with itsinteraction partners (e.g. adaptor molecules). This can be performed,for example, with the FRET (frequency resonance energy transfer) systemby 9B5 being expressed in fusion and purified with GFP (greenfluorescent protein) and its interaction partner with BFP (bluefluorescent protein). In a cell-free system, the reduction in emissionof BFP can then be used as an indicator of the presence of an inhibitorwhen searching complex chemical banks.

[0122] The main aim behind therapeutic exploitation of the proteinkinase detected is, however, firstly to eliminate the protein kinasefunction, as this can be performed most easily. Protein kinases presentthemselves in principle for the performance of high-throughput assaysfor the identification of inhibitors (small-molecule inhibitors) of thekinase property of the protein as the enzyme property itself can bereadily used as indicator (see also example 3, kinase assays).

[0123] Simple implementation of an HTS system for 9B5 can be performedby the filter assay method of Reuter et al. (Reuter et al., Methods inEnzymology, 255: 245 (1995)), with MBP being used as non-specificsubstrate. This is applied to 96-hole plates that are suitable for ELISA(e.g. FlashPlates, NEN Life Science Products, or NUNC). Reaction buffer(3× kinase reaction buffer contains: 60 mM HEPES (pH 7.5), 30 mM MgAc,0.15 mM gammaATP, 3 mM DTT, 0.03 mM Na-orthovanadate) is added. 0.25 μCi33P-gamma-ATP and the kinase described are added in a concentration ofno more than 1 μg/ml (a titration should first be performed). In thepresence of the potential inhibitor (e.g. small molecules from achemical bank) (e.g. 10 μM), the reaction is incubated at 30° for 1 h.The total reaction volume is 100 μl. The reaction is stopped by theaddition of EDTA (pH 7) up to a final concentration of 80 mM. Thesamples are then centrifuged, and 50 μl of the supernatant is applied top81 cation exchanger paper (manufactured by Whatman). The filters arethen washed 3 times in 200 ml 180 mM phosphoric acid (every 5 mins), andonce in 200 ml 96% ethanol. Following drying in air, the radioactivityof the filters is determined by scintillation counting. Substances thatreduce kinase activity by >=50% (at 10 μM) stand out as a result ofa >50% reduction in incorporation. The specificity and sensitivity ofthe possible inhibitors are determined by titration to determine IC50,and by substitution of other kinases in the assay. Relative comparisonsof the inhibition effect on other kinases thus allow for a measure ofspecificity.

[0124] For the performance of screens on kinase inhibitors, more modernsystems with the scintillation proximity assay (SPA) also presentthemselves (manufactured by Amersham Life Science, MAP kinase SPA;(Zhang et al., Proc Natl Acad Sci USA, 96, 8511-5, (1999)), (Ichijo etal., Science, 275, 90-4, (1997))). McDonald describes an assay set-upfor the Raf/MEK/ERK cascade that can identify inhibitors of the entirecascade. A biotinylated peptide which, following phosphorylation with³³P, can bind to avidin-coated SPA beads is used here. The MAP cascadeis here reconstituted in vitro, expressed with the individualconstituents as GST fusion protein in E. coli or, in the case of cRAF1,produced with the Baculovirus system. The first element of the cascade(MAP-KKK) must in this connection always be uniformly activated to beable to screen inhibitors reliably. This was achieved in this case bycoexpression of src in the Baculovirus system. A ras-analogousactivation of cRaf is thereby achieved. Another way of activating a MAPkinase by phosphorylation is also conceivable in principle. Anotherpossibility of constitutive activation of a MAP kinase consists inmutation of the amino acids to be phosphorylated. This was, for example,possible in the case of MEK1 via the replacement of the serines Ser218and Ser222 by glutamate (Zhang et al., Proc Natl Acad Sci USA, 96,8511-5, (1999)), (Ichijo et al., Science, 275, 90-4, (1997)). In thecase of 9B5, an interaction screen (e.g. y2h system) can firstly beperformed to identify a phosphorylation target or MBP (myelin basicprotein) directly used as substrate. A peptide from the target sequencecan then be synthesised with a biotin anchor. This can bind toavidin-coupled SPA beads manufactured by Amersham. Purified (e.g.bacterially produced) 9B5 protein and gamma-32P-ATP are still addedduring the reaction. This can take place on a microtitre scale. Undernormal conditions, the target peptide is phosphorylated and will triggera scintillation response. From a library of chemical substances, thosewith the reaction will now be pre-incubated prior to addition of thetarget peptide. Following addition of the target peptide, thescintillation response can then be measured. A drop in responsesignifies the presence of a potential inhibitor. The library searchedshould be as complex as possible, and contain many different substanceclasses. Examples of a substance class-specific inhibitor for a proteinkinase (p38) can be found for example in U.S. Pat. No. 5,945,418. Othersubstance classes that can inhibit protein kinases are for example bismonocyclic, bicyclic or heterocyclic aryl compounds (WO 92/20642),vinylene-azaindol derivatives (WO 94/14808),1-cyclopropyl-4-pyridyl-quinolone (U.S. Pat. No. 5,330,992), styrylcompounds (U.S. Pat. No. 5,217,999), styryl-substituted pyridylcompounds (U.S. Pat. No. 5,302,606), certain quinazoline derivatives (EPApplication No. 0 566 266 A1), Selenoindoles and selenides (WO94/03427), tricyclic polyhydroxylic compounds (WO 92/21660), andbenzylphosphonic acids (WO 91/15495).

[0125] The specific nature of the assay is solely determined by theidentity of the Ser/Thr kinase described. Various substrates can beused. These may be used in various forms of the aforementioned assayprinciple. The essential point in this is, however, always the use ofthe kinase activity of 9B5 for screening purposes. For example, thesubstrate may occur in free solution as well as being coupled to aphase, as mentioned above. The substrate may, however, also be borne ona cell surface or even be presented intracellularly. This does notaffect the underlying assay principle.

Example 7 Production of Transgenic and Knock-Out Mice

[0126] Knowledge of the sequence of 9B5 can be used to producegenetically modified mice (or other animals). To this end, for example aconstitutively inactive mutant of 9B5 is expressed in transgenic mice,e.g. with an NSE promoter in neurons, with an MBP promoter inoligodendrocytes, etc. This should have a dominant-negative effect, andthus imitate inhibition of 9B5. This may yield valuable indications ofpossible pharmacological effects of inhibitors in vivo. A constitutivelyactive kinase can also be expressed.

[0127] The production of knock-out animals may also yield indications ofthe effects of inhibitors. The production of these genetically modifiedmice or other animals takes place in accordance with standard methodsfamiliar to the expert.

[0128] The genetically modified animals can then be investigated invarious disease models (e.g. experimentally induced stroke, MCAO), ortumour models.

Example 8 Proliferation Assays

[0129] Many of the previously known serine threonine kinases areinvolved in neoplastic processes. The involvement of 9B5 in the growthof cells, the cell cycle and tumorigenic transformation can be furtherinvestigated by transfecting expression constructs with 9B5 ineukaryotic cells and then investigating the induction of tumorigenicity,e.g. in a Soft-Agar test (Zhang et al., Proc Natl Acad Sci USA, 96,8511-5, (1999)), (Ichijo et al., Science, 275, 90-4, (1997)). On celltypes, several customary lines, e.g. Cos cells, HEK cells, PC12 cells,THP-1 cells and primary cells, such as for example neurons andastrocytes, are considered, as are other immortalised and primary celllines, as required.

1 8 1 3170 DNA Mus musculus polyA_signal (3094)..(3099) Coding sequence(1)...(2172) 1 aaagggccgt cctggtccag ccgttccctg ggtgcccgtt gccggaactctatcgcttcc 60 tgccctgagg aacaacccca tgtgggcaac tataggctgc taaggaccatcgggaagggc 120 aacttcgcca aagtcaagct ggctcggcat atcctcacgg gccgggaggtcgctattaag 180 atcattgata agacccagct gaaccccagt agcttgcaga agctgttcagagaagtccga 240 attatgaagg gactcaacca ccccaacatc gtgaagcttt ttgaggtgatagagacggag 300 aagacgctat acctggtgat ggaatacgct agcgcaggag aagtgtttgactacctcgtg 360 tcgcacggcc gcatgaagga gaaggaggct cgagccaagt tgcggcagatcgtgtcagcc 420 gtgcactact gtcatcagaa gaacattgta cacagggatc taaaggctgaaaacctgttg 480 ctggatgccg aggccaacat caaaatcgcc gacttcggct tcagcaatgagttcacgctg 540 ggctccaagc tggacacctt ctgtgggagc cccccatacg ccgccccagagctgttccag 600 ggcaagaagt atgatgggcc agaggtggac atctggagcc tgggtgtcatcctgtacacg 660 ctggtcagcg gctccctgcc cttcgatggg cacaacctca aggagctgcgggagcgaatc 720 ctcagaggaa agtaccgggt ccccttctac atgtctacag actgcgagagcattctgcgg 780 agatttctgg tgctgaaccc cgcaaaacgc tgtactctgg agcaaatcatgaaagacaaa 840 tggatcaaca tcggctatga gggtgaggag ctgaagccat acacggagcctgaggaggac 900 ttcggggaca ccaagagaat tgaggtgatg gtgggtatgg gctacacacgggaagaaatc 960 aaagaggcct tgaccaacca gaagtacaac gaggtgaccg ccacctacctcctgctgggc 1020 aggaagactg atgagggtgg ggaccggggt gccccagggc tggccctggcacgggtgcgg 1080 gcgcccagcg acaccaccaa cgggacaagc tccagcaaag gcagcagccacaacaaaggg 1140 caacgggctt cttcctccac ctaccaccgc cagcgccgtc acagtgacttctgtggcccg 1200 tcccctgccc cgctgcaccc gaagcgcacg ccaaccagca cgggagacacggagctcaaa 1260 gaagagcgga tgccgggtcg gaaagcgagc tgcagtgcag tgggcagtggaagtcgaggc 1320 ttgcccccct ccagccccat ggtcagcagt gcccacaacc ccaataaggcagagatccct 1380 gagcggcgga aggacagcac tagcacccct aacaacctcc cccccagcatgatgacccga 1440 agaaacacct atgtgtgcac agagcgacca ggatctgaac gcccgtccttgttgccaaat 1500 ggcaaagaaa atagctccgg tacctcgcgg gtgccccctg cctcgccttccagtcatagc 1560 ctggctcccc cgtcaggcga gcggagccgc ctggctcggg gctccaccatccgcagcacc 1620 ttccatgggg gccaggtccg agaccggcgg gcagggagcg ggagtggcgggggtgtgcag 1680 aatggacccc cagcctcacc cacgcttgcc cacgaggccg cacccctgccctccgggcgg 1740 cctcgcccca ccaccaacct cttcaccaag ctgacctcca aactgacccgaagggtcaca 1800 gacgaacctg agagaatcgg gggacctgag gtcacaagtt gccatctaccttgggataaa 1860 acggaaaccg cccccaggct gctccgattc ccctggagtg tgaagctgaccagctcgcga 1920 cctcctgagg ccctgatggc tgccatgcga caggccacag cggccgcccgctgccggtgc 1980 cgccagccgc agccgttcct gctggcctgc ctgcacgggg gtgcgggcgggcccgagccc 2040 ctgtcccatt tcgaagtgga ggtgtgccag ctgccccggc ccggcctcaggggcgtcctc 2100 ttccgccgtg tggcgggcac cgccctggcc ttccgcaccc tcgtcacccgcatctccaac 2160 gacctcgaac tctgagccac cgccaccact accaccgcca cagccaccatcacagcccgg 2220 gtcccttctt tctctggttc ctttcacttc cccaagaggg gaagaggacagaggagaggg 2280 tgccctgtgt catgactgaa gtttccctgg attagattgg tggacagagacagtgtgggg 2340 acacatgaca tgataagagg gctcagcagg gggagctggc accctcctagggcctctggt 2400 gggacccccc tccccacaat cttgttcttc tgcagggcac ctgaggagactttggggaca 2460 ggagtgagaa gggaaactga ggaaattctc ccattcaggg agagctgccaggattaatga 2520 ctggagacag acttgggggg ttcagggagt tgggggagtc acagacagaaaccttcccct 2580 cactccccct tatgatcgaa cctccttcat gccccaggct ggcgcggggcactttgtaca 2640 aatccgtgta tatactcctg tccctctgca gaggtctctc ggggagctgctgctgccgcc 2700 tccgattttt aagttattgc cccgcccctt ctgtcagctc ctcatctgcagcctgttact 2760 caataaacag taggagtccc tccaaccccg acctcctccc tggccgacctggggtttccc 2820 ttctcagccc ttggcctgca ggtgagccag ggagctgggg acttgaccccaacctgtggt 2880 tctgcttgct gagcctttgt tatctcatct tcagaatggg aacagtggggttggaggatg 2940 ggtcaaggat gactatggaa gagggcagaa cagagctcag cctcttccacgaggccccag 3000 ccttctgtga caccctcctc ttggccactc actcccctct gccatattacactggaccca 3060 gagcctcttc ctattccagt aatacatgta ttcaataaac aatcaacgactggtgccgac 3120 tccacgctag gcccagttct ggacataaaa aaaaaaaaaa aaaaaaaaaa3170 2 3250 DNA Mus musculus polyA_signal (3174)..(3179) Coding sequence(1)...(1980) 2 aaagggccgt cctggtccag ccgttccctg ggtgcccgtt gccggaactctatcgcttcc 60 tgccctgagg aacaacccca tgtgggcaac tataggctgc taaggaccatcgggaagggc 120 aacttcgcca aagtcaagct ggctcggcat atcctcacgg gccgggaggtcgctattaag 180 atcattgata agacccagct gaaccccagt agcttgcaga agctgttcagagaagtccga 240 attatgaagg gactcaacca ccccaacatc gtgaagcttt ttgaggtgatagagacggag 300 aagacgctat acctggtgat ggaatacgct agcgcaggag aagtgtttgactacctcgtg 360 tcgcacggcc gcatgaagga gaaggaggct cgagccaagt tgcggcagatcgtgtcagcc 420 gtgcactact gtcatcagaa gaacattgta cacagggatc taaaggctgaaaacctgttg 480 ctggatgccg aggccaacat caaaatcgcc gacttcggct tcagcaatgagttcacgctg 540 ggctccaagc tggacacctt ctgtgggagc cccccatacg ccgccccagagctgttccag 600 ggcaagaagt atgatgggcc agaggtggac atctggagcc tgggtgtcatcctgtacacg 660 ctggtcagcg gctccctgcc cttcgatggg cacaacctca aggagctgcgggagcgaatc 720 ctcagaggaa agtaccgggt ccccttctac atgtctacag actgcgagagcattctgcgg 780 agatttctgg tgctgaaccc cgcaaaacgc tgtactctgg agcaaatcatgaaagacaaa 840 tggatcaaca tcggctatga gggtgaggag ctgaagccat acacggagcctgaggaggac 900 ttcggggaca ccaagagaat tgaggtgatg gtgggtatgg gctacacacgggaagaaatc 960 aaagaggcct tgaccaacca gaagtacaac gaggtgaccg ccacctacctcctgctgggc 1020 aggaagactg atgagggtgg ggaccggggt gccccagggc tggccctggcacgggtgcgg 1080 gcgcccagcg acaccaccaa cgggacaagc tccagcaaag gcagcagccacaacaaaggg 1140 caacgggctt cttcctccac ctaccaccgc cagcgccgtc acagtgacttctgtggcccg 1200 tcccctgccc cgctgcaccc gaagcgcagc ccaaccagca cgggagacacggagctcaaa 1260 gaagagcgga tgccgggtcg gaaagcgagc tgcagtgcag tgggcagtggaagtcgaggc 1320 ttgcccccct ccagccccat ggtcagcagt gcccacaacc ccaataaggcagagatccct 1380 gagcggcgga aggacagcac tagcacccct aacaacctcc cccccagcatgatgacccga 1440 agaaacacct atgtgtgcac agagcgacca ggatctgaac gcccgtccttgttgccaaat 1500 ggcaaagaaa atagctccgg tacctcgcgg gtgccccctg cctcgccttccagtcatagc 1560 ctggctcccc cgtcaggcga gcggagccgc ctggctcggg gctccaccatccgcagcacc 1620 ttccatgggg gccaggtccg agaccggcgg gcagggagcg ggagtggcgggggtgtgcag 1680 aatggacccc cagcctcacc cacgcttgcc cacgaggccg cacccctgccctccgggcgg 1740 cctcgcccca ccaccaacct cttcaccaag ctgacctcca aactgacccgaagggttacc 1800 ctcgatccct ctaaacggca gaactctaac cgctgtgtct cgggcgcctctctgccccag 1860 ggatccaaaa tcaggtcaca gacgaacctg agagaatcgg gggacctgaggtcacaagtt 1920 gccatctacc ttgggataaa acggaaaccg cccccaggct gctccgattcccctggagtg 1980 tgaagctgac cagctcgcga cctcctgagg ccctgatggc tgccatgcgacaggccacag 2040 cggccgcccg ctgccggtgc cgccagccgc agccgttcct gctggcctgcctgcacgggg 2100 gtgcgggcgg gcccgagccc ctgtcccatt tcgaagtgga ggtgtgccagctgccccggc 2160 ccggcctcag gggcgtcctc ttccgccgtg tggcgggcac cgccctggccttccgcaccc 2220 tcgtcacccg catctccaac gacctcgaac tctgagccac cgccaccactaccaccgcca 2280 cagccaccat cacagcccgg gtcccttctt tctctggttc ctttcacttccccaagaggg 2340 gaagaggaca gaggagaggg tgccctgtgt catgactgaa gtttccctggattagattgg 2400 tggacagaga cagtgtgggg acacatgaca tgataagagg gctcagcagggggagctggc 2460 accctcctag ggcctctggt gggacccccc tccccacaat cttgttcttctgcagggcac 2520 ctgaggagac tttggggaca ggagtgagaa gggaaactga ggaaattctcccattcaggg 2580 agagctgcca ggattaatga ctggagacag acttgggggg ttcagggagttgggggagtc 2640 acagacagaa accttcccct cactccccct tatgatcgaa cctccttcatgccccaggct 2700 ggcgcggggc actttgtaca aatccgtgta tatactcctg tccctctgcagaggtctctc 2760 ggggagctgc tgctgccgcc tccgattttt aagttattgc cccgccccttctgtcagctc 2820 ctcatctgca gcctgttact caataaacag taggagtccc tccaaccccgacctcctccc 2880 tggccgacct ggggtttccc ttctcagccc ttggcctgca ggtgagccagggagctgggg 2940 acttgacccc aacctgtggt tctgcttgct gagcctttgt tatctcatcttcagaatggg 3000 aacagtgggg ttggaggatg ggtcaaggat gactatggaa gagggcagaacagagctcag 3060 cctcttccac gaggccccag ccttctgtga caccctcctc ttggccactcactcccctct 3120 gccatattac actggaccca gagcctcttc ctattccagt aatacatgtattcaataaac 3180 aatcaacgac tggtgccgac tccacgctag gcccagttct ggacataaaaaaaaaaaaaa 3240 aaaaaaaaaa 3250 3 3312 DNA Homo sapiens polyA_signal(3275)..(3280) Coding sequence (64)...(2319) 3 aggggaccct gggacccccgccccccccac ccggccgccc ctgccccccg ggacccggag 60 aagatgtctt cgcggacggtgctggccccg ggcaacgatc ggaactcgga cacgcatggc 120 accttgggca gtggccgctcctcggacaaa ggcccgtcct ggtccagccg ctcactgggt 180 gcccgttgcc ggaactccatcgcctcctgt cccgaggagc agccccacgt gggcaactac 240 cgcctgctga ggaccattgggaagggcaac tttgccaaag tcaagctggc tcggcacatc 300 ctcactggtc gggaggttgccatcaagatt atcgacaaaa cccagctgaa tcccagcagc 360 ctgcagaagc tgttccgagaagtccgcatc atgaagggcc taaaccaccc caacatcgtg 420 aagctctttg aggtgattgagactgagaag acgctgtacc tggtgatgga gtacgcaagt 480 gctggagaag tgtttgactacctcgtgtcg catggccgca tgaaggagaa ggaagctcga 540 gccaagttcc gacagattgtttcggctgtg cactattgtc accagaaaaa tattgtacac 600 agggacctga aggctgagaacctcttgctg gatgccgagg ccaacatcaa gattgctgac 660 tttggcttca gcaacgagttcacgctggga tcgaagctgg acacgttctg cgggagcccc 720 ccatatgccg ccccggagctgtttcagggc aagaagtacg acgggccgga ggtggacatc 780 tggagcctgg gagtcatcctgtacaccctc gtcagcggct ccctgccctt cgacgggcac 840 aacctcaagg agctgcgggagcgagtactc agagggaagt accgggtccc tttctacatg 900 tcaacagact gtgagagcatcctgcggaga tttttggtgc tgaacccagc taaacgctgt 960 actctcgagc aaatcatgaaagacaaatgg atcaacatcg gctatgaggg tgaggagttg 1020 aagccataca cagagcccgaggaggacttc ggggacacca agagaattga ggtgatggtg 1080 ggtatgggct acacacgggaagaaatcaaa gagtccttga ccagccagaa gtacaacgaa 1140 gtgaccgcca cctacctcctgctgggcagg aagactgagg agggtgggga ccggggcgcc 1200 ccagggctgg ccctggcacgggtgcgggcg cccagcgaca ccaccaacgg aacaagttcc 1260 agcaaaggca ccagccacagcaaagggcag cggagttcct cttccaccta ccaccgccag 1320 cgcaggcata gcgatttctgtggcccatcc cctgcacccc tgcaccccaa acgcagcccg 1380 acgagcacgg gggaggcggagctgaaggag gagcggctgc caggccggaa ggcgagctgc 1440 agcaccgcgg ggagtgggagtcgagggctg cccccctcca gccccatggt cagcagcgcc 1500 cacaacccca acaaggcagagatcccagag cggcggaagg acagcacgag cacccccaac 1560 aacctccctc ctagcatgatgacccgcaga aacacctacg tttgcacaga acgcccgggg 1620 gctgagcgcc cgtcactgttgccaaatggg aaagaaaaca gctcaggcac cccacgggtg 1680 ccccctgcct ccccctccagtcacagcctg gcacccccat caggggagcg gagccgcctg 1740 gcacgtggtt ccaccatccgcagcaccttc catggtggcc aggtccggga ccggcgggca 1800 gggggtgggg gtggtgggggtgtgcagaat gggccccctg cctctcccac actggcccat 1860 gaggctgcac ccctgcccgccgggcggccc cgccccacca ccaacctctt caccaagctg 1920 acctccaaac tgacccgaagggtcgcagac gaacctgaga gaatcggggg acctgaggtc 1980 acaagttgcc atctaccttgggatcaaacg gaaaccgccc cccggctgct ccgattcccc 2040 tggagtgtga agctgaccagctcgcgccct cctgaggccc tgatggcagc tctgcgccag 2100 gccacagcag ccgcccgctgccgctgccgc cagccacagc cgttcctgct ggcctgcctg 2160 cacgggggtg cgggcgggcccgagcccctg tcccacttcg aagtggaggt ctgccagctg 2220 ccccggccag gcttgcggggagttctcttc cgccgtgtgg cgggcaccgc cctggccttc 2280 cgcaccctcg tcacccgcatctccaacgac ctcgagctct gagccaccac ggtcccaggg 2340 cccttactct tcctctcccttgtcgccttc acttctacag gaggggaagg ggccagggag 2400 gggattctcc ctttatcatcacctcagttt ccctgaatta tatttggggg caaagattgt 2460 cccctctgct gttctctggggccgctcagc acagaagaag gatgaggggg ctcagcgggg 2520 ggagctggca ccttcctggagcctccagcc agtcctgtcc tccctcgccc taccaagagg 2580 gcacctgagg agactttggggacagggcag gggcagggag ggaaactgag gaaatcttcc 2640 attcctccca acagctcaaaattaggcctt gggcaggggc agggagagct gctgagccta 2700 aagactggag aatctgggggactgggagtg ggggtcagag aggcagattc cttcccctcc 2760 cgtcccctca cgctcaaacccccacttcct gccccaggct ggcgcggggc actttgtaca 2820 aatccttgta aataccccacaccctcccct ctgcaaaggt ctcttgagga gctgccgctg 2880 tcacctacgg tttttaagttattacacccc gaccctcctc ctgtcagccc cctcacctgc 2940 agcctgttgc ccaataaatttaggagagtc cccccctccc caatgctgac cctaggattt 3000 tccttccctg ccctcacctgcaaatgagtt aaagaagagg cgtgggaatc caggcagtgg 3060 tttttccttt cggagcctcggttttctcat ctgcagaatg ggagcggtgg gggtgggaag 3120 gtaaggatgg tcgtggaagaaggcaggatg gaactcggcc tcatccccga ggccccagtt 3180 cctatatcgg gccccccattcatccactca cactcccagc caccatgtta cactggactc 3240 taagccactt cttactccagtagtaaattt attcaataaa caatcattga cccatgccta 3300 aaaaaaaaaa aa 3312 43392 DNA Homo sapiens polyA_signal (3355)..(3360) Coding sequence(64)...(2127) 4 aggggaccct gggacccccg ccccccccac ccggccgccc ctgccccccgggacccggag 60 aagatgtctt cgcggacggt gctggccccg ggcaacgatc ggaactcggacacgcatggc 120 accttgggca gtggccgctc ctcggacaaa ggcccgtcct ggtccagccgctcactgggt 180 gcccgttgcc ggaactccat cgcctcctgt cccgaggagc agccccacgtgggcaactac 240 cgcctgctga ggaccattgg gaagggcaac tttgccaaag tcaagctggctcggcacatc 300 ctcactggtc gggaggttgc catcaagatt atcgacaaaa cccagctgaatcccagcagc 360 ctgcagaagc tgttccgaga agtccgcatc atgaagggcc taaaccaccccaacatcgtg 420 aagctctttg aggtgattga gactgagaag acgctgtacc tggtgatggagtacgcaagt 480 gctggagaag tgtttgacta cctcgtgtcg catggccgca tgaaggagaaggaagctcga 540 gccaagttcc gacagattgt ttcggctgtg cactattgtc accagaaaaatattgtacac 600 agggacctga aggctgagaa cctcttgctg gatgccgagg ccaacatcaagattgctgac 660 tttggcttca gcaacgagtt cacgctggga tcgaagctgg acacgttctgcgggagcccc 720 ccatatgccg ccccggagct gtttcagggc aagaagtacg acgggccggaggtggacatc 780 tggagcctgg gagtcatcct gtacaccctc gtcagcggct ccctgcccttcgacgggcac 840 aacctcaagg agctgcggga gcgagtactc agagggaagt accgggtccctttctacatg 900 tcaacagact gtgagagcat cctgcggaga tttttggtgc tgaacccagctaaacgctgt 960 actctcgagc aaatcatgaa agacaaatgg atcaacatcg gctatgagggtgaggagttg 1020 aagccataca cagagcccga ggaggacttc ggggacacca agagaattgaggtgatggtg 1080 ggtatgggct acacacggga agaaatcaaa gagtccttga ccagccagaagtacaacgaa 1140 gtgaccgcca cctacctcct gctgggcagg aagactgagg agggtggggaccggggcgcc 1200 ccagggctgg ccctggcacg ggtgcgggcg cccagcgaca ccaccaacggaacaagttcc 1260 agcaaaggca ccagccacag caaagggcag cggagttcct cttccacctaccaccgccag 1320 cgcaggcata gcgatttctg tggcccatcc cctgcacccc tgcaccccaaacgcagcccg 1380 acgagcacgg gggaggcgga gctgaaggag gagcggctgc caggccggaaggcgagctgc 1440 agcaccgcgg ggagtgggag tcgagggctg cccccctcca gccccatggtcagcagcgcc 1500 cacaacccca acaaggcaga gatcccagag cggcggaagg acagcacgagcacccccaac 1560 aacctccctc ctagcatgat gacccgcaga aacacctacg tttgcacagaacgcccgggg 1620 gctgagcgcc cgtcactgtt gccaaatggg aaagaaaaca gctcaggcaccccacgggtg 1680 ccccctgcct ccccctccag tcacagcctg gcacccccat caggggagcggagccgcctg 1740 gcacgtggtt ccaccatccg cagcaccttc catggtggcc aggtccgggaccggcgggca 1800 gggggtgggg gtggtggggg tgtgcagaat gggccccctg cctctcccacactggcccat 1860 gaggctgcac ccctgcccgc cgggcggccc cgccccacca ccaacctcttcaccaagctg 1920 acctccaaac tgacccgaag ggttaccctc gatccctcta aacggcagaactctaaccgc 1980 tgtgtttcgg gcgcctctct gccccaggga tccaagatca ggtcgcagacgaacctgaga 2040 gaatcggggg acctgaggtc acaagttgcc atctaccttg ggatcaaacggaaaccgccc 2100 cccggctgct ccgattcccc tggagtgtga agctgaccag ctcgcgccctcctgaggccc 2160 tgatggcagc tctgcgccag gccacagcag ccgcccgctg ccgctgccgccagccacagc 2220 cgttcctgct ggcctgcctg cacgggggtg cgggcgggcc cgagcccctgtcccacttcg 2280 aagtggaggt ctgccagctg ccccggccag gcttgcgggg agttctcttccgccgtgtgg 2340 cgggcaccgc cctggccttc cgcaccctcg tcacccgcat ctccaacgacctcgagctct 2400 gagccaccac ggtcccaggg cccttactct tcctctccct tgtcgccttcacttctacag 2460 gaggggaagg ggccagggag gggattctcc ctttatcatc acctcagtttccctgaatta 2520 tatttggggg caaagattgt cccctctgct gttctctggg gccgctcagcacagaagaag 2580 gatgaggggg ctcagcgggg ggagctggca ccttcctgga gcctccagccagtcctgtcc 2640 tccctcgccc taccaagagg gcacctgagg agactttggg gacagggcaggggcagggag 2700 ggaaactgag gaaatcttcc attcctccca acagctcaaa attaggccttgggcaggggc 2760 agggagagct gctgagccta aagactggag aatctggggg actgggagtgggggtcagag 2820 aggcagattc cttcccctcc cgtcccctca cgctcaaacc cccacttcctgccccaggct 2880 ggcgcggggc actttgtaca aatccttgta aataccccac accctcccctctgcaaaggt 2940 ctcttgagga gctgccgctg tcacctacgg tttttaagtt attacaccccgaccctcctc 3000 ctgtcagccc cctcacctgc agcctgttgc ccaataaatt taggagagtccccccctccc 3060 caatgctgac cctaggattt tccttccctg ccctcacctg caaatgagttaaagaagagg 3120 cgtgggaatc caggcagtgg tttttccttt cggagcctcg gttttctcatctgcagaatg 3180 ggagcggtgg gggtgggaag gtaaggatgg tcgtggaaga aggcaggatggaactcggcc 3240 tcatccccga ggccccagtt cctatatcgg gccccccatt catccactcacactcccagc 3300 caccatgtta cactggactc taagccactt cttactccag tagtaaatttattcaataaa 3360 caatcattga cccatgccta aaaaaaaaaa aa 3392 5 724 PRT Musmusculus 5 Lys Gly Pro Ser Trp Ser Ser Arg Ser Leu Gly Ala Arg Cys ArgAsn 1 5 10 15 Ser Ile Ala Ser Cys Pro Glu Glu Gln Pro His Val Gly AsnTyr Arg 20 25 30 Leu Leu Arg Thr Ile Gly Lys Gly Asn Phe Ala Lys Val LysLeu Ala 35 40 45 Arg His Ile Leu Thr Gly Arg Glu Val Ala Ile Lys Ile IleAsp Lys 50 55 60 Thr Gln Leu Asn Pro Ser Ser Leu Gln Lys Leu Phe Arg GluVal Arg 65 70 75 80 Ile Met Lys Gly Leu Asn His Pro Asn Ile Val Lys LeuPhe Glu Val 85 90 95 Ile Glu Thr Glu Lys Thr Leu Tyr Leu Val Met Glu TyrAla Ser Ala 100 105 110 Gly Glu Val Phe Asp Tyr Leu Val Ser His Gly ArgMet Lys Glu Lys 115 120 125 Glu Ala Arg Ala Lys Leu Arg Gln Ile Val SerAla Val His Tyr Cys 130 135 140 His Gln Lys Asn Ile Val His Arg Asp LeuLys Ala Glu Asn Leu Leu 145 150 155 160 Leu Asp Ala Glu Ala Asn Ile LysIle Ala Asp Phe Gly Phe Ser Asn 165 170 175 Glu Phe Thr Leu Gly Ser LysLeu Asp Thr Phe Cys Gly Ser Pro Pro 180 185 190 Tyr Ala Ala Pro Glu LeuPhe Gln Gly Lys Lys Tyr Asp Gly Pro Glu 195 200 205 Val Asp Ile Trp SerLeu Gly Val Ile Leu Tyr Thr Leu Val Ser Gly 210 215 220 Ser Leu Pro PheAsp Gly His Asn Leu Lys Glu Leu Arg Glu Arg Ile 225 230 235 240 Leu ArgGly Lys Tyr Arg Val Pro Phe Tyr Met Ser Thr Asp Cys Glu 245 250 255 SerIle Leu Arg Arg Phe Leu Val Leu Asn Pro Ala Lys Arg Cys Thr 260 265 270Leu Glu Gln Ile Met Lys Asp Lys Trp Ile Asn Ile Gly Tyr Glu Gly 275 280285 Glu Glu Leu Lys Pro Tyr Thr Glu Pro Glu Glu Asp Phe Gly Asp Thr 290295 300 Lys Arg Ile Glu Val Met Val Gly Met Gly Tyr Thr Arg Glu Glu Ile305 310 315 320 Lys Glu Ala Leu Thr Asn Gln Lys Tyr Asn Glu Val Thr AlaThr Tyr 325 330 335 Leu Leu Leu Gly Arg Lys Thr Asp Glu Gly Gly Asp ArgGly Ala Pro 340 345 350 Gly Leu Ala Leu Ala Arg Val Arg Ala Pro Ser AspThr Thr Asn Gly 355 360 365 Thr Ser Ser Ser Lys Gly Ser Ser His Asn LysGly Gln Arg Ala Ser 370 375 380 Ser Ser Thr Tyr His Arg Gln Arg Arg HisSer Asp Phe Cys Gly Pro 385 390 395 400 Ser Pro Ala Pro Leu His Pro LysArg Thr Pro Thr Ser Thr Gly Asp 405 410 415 Thr Glu Leu Lys Glu Glu ArgMet Pro Gly Arg Lys Ala Ser Cys Ser 420 425 430 Ala Val Gly Ser Gly SerArg Gly Leu Pro Pro Ser Ser Pro Met Val 435 440 445 Ser Ser Ala His AsnPro Asn Lys Ala Glu Ile Pro Glu Arg Arg Lys 450 455 460 Asp Ser Thr SerThr Pro Asn Asn Leu Pro Pro Ser Met Met Thr Arg 465 470 475 480 Arg AsnThr Tyr Val Cys Thr Glu Arg Pro Gly Ser Glu Arg Pro Ser 485 490 495 LeuLeu Pro Asn Gly Lys Glu Asn Ser Ser Gly Thr Ser Arg Val Pro 500 505 510Pro Ala Ser Pro Ser Ser His Ser Leu Ala Pro Pro Ser Gly Glu Arg 515 520525 Ser Arg Leu Ala Arg Gly Ser Thr Ile Arg Ser Thr Phe His Gly Gly 530535 540 Gln Val Arg Asp Arg Arg Ala Gly Ser Gly Ser Gly Gly Gly Val Gln545 550 555 560 Asn Gly Pro Pro Ala Ser Pro Thr Leu Ala His Glu Ala AlaPro Leu 565 570 575 Pro Ser Gly Arg Pro Arg Pro Thr Thr Asn Leu Phe ThrLys Leu Thr 580 585 590 Ser Lys Leu Thr Arg Arg Val Thr Asp Glu Pro GluArg Ile Gly Gly 595 600 605 Pro Glu Val Thr Ser Cys His Leu Pro Trp AspLys Thr Glu Thr Ala 610 615 620 Pro Arg Leu Leu Arg Phe Pro Trp Ser ValLys Leu Thr Ser Ser Arg 625 630 635 640 Pro Pro Glu Ala Leu Met Ala AlaMet Arg Gln Ala Thr Ala Ala Ala 645 650 655 Arg Cys Arg Cys Arg Gln ProGln Pro Phe Leu Leu Ala Cys Leu His 660 665 670 Gly Gly Ala Gly Gly ProGlu Pro Leu Ser His Phe Glu Val Glu Val 675 680 685 Cys Gln Leu Pro ArgPro Gly Leu Arg Gly Val Leu Phe Arg Arg Val 690 695 700 Ala Gly Thr AlaLeu Ala Phe Arg Thr Leu Val Thr Arg Ile Ser Asn 705 710 715 720 Asp LeuGlu Leu 6 660 PRT Mus musculus 6 Lys Gly Pro Ser Trp Ser Ser Arg Ser LeuGly Ala Arg Cys Arg Asn 1 5 10 15 Ser Ile Ala Ser Cys Pro Glu Glu GlnPro His Val Gly Asn Tyr Arg 20 25 30 Leu Leu Arg Thr Ile Gly Lys Gly AsnPhe Ala Lys Val Lys Leu Ala 35 40 45 Arg His Ile Leu Thr Gly Arg Glu ValAla Ile Lys Ile Ile Asp Lys 50 55 60 Thr Gln Leu Asn Pro Ser Ser Leu GlnLys Leu Phe Arg Glu Val Arg 65 70 75 80 Ile Met Lys Gly Leu Asn His ProAsn Ile Val Lys Leu Phe Glu Val 85 90 95 Ile Glu Thr Glu Lys Thr Leu TyrLeu Val Met Glu Tyr Ala Ser Ala 100 105 110 Gly Glu Val Phe Asp Tyr LeuVal Ser His Gly Arg Met Lys Glu Lys 115 120 125 Glu Ala Arg Ala Lys LeuArg Gln Ile Val Ser Ala Val His Tyr Cys 130 135 140 His Gln Lys Asn IleVal His Arg Asp Leu Lys Ala Glu Asn Leu Leu 145 150 155 160 Leu Asp AlaGlu Ala Asn Ile Lys Ile Ala Asp Phe Gly Phe Ser Asn 165 170 175 Glu PheThr Leu Gly Ser Lys Leu Asp Thr Phe Cys Gly Ser Pro Pro 180 185 190 TyrAla Ala Pro Glu Leu Phe Gln Gly Lys Lys Tyr Asp Gly Pro Glu 195 200 205Val Asp Ile Trp Ser Leu Gly Val Ile Leu Tyr Thr Leu Val Ser Gly 210 215220 Ser Leu Pro Phe Asp Gly His Asn Leu Lys Glu Leu Arg Glu Arg Ile 225230 235 240 Leu Arg Gly Lys Tyr Arg Val Pro Phe Tyr Met Ser Thr Asp CysGlu 245 250 255 Ser Ile Leu Arg Arg Phe Leu Val Leu Asn Pro Ala Lys ArgCys Thr 260 265 270 Leu Glu Gln Ile Met Lys Asp Lys Trp Ile Asn Ile GlyTyr Glu Gly 275 280 285 Glu Glu Leu Lys Pro Tyr Thr Glu Pro Glu Glu AspPhe Gly Asp Thr 290 295 300 Lys Arg Ile Glu Val Met Val Gly Met Gly TyrThr Arg Glu Glu Ile 305 310 315 320 Lys Glu Ala Leu Thr Asn Gln Lys TyrAsn Glu Val Thr Ala Thr Tyr 325 330 335 Leu Leu Leu Gly Arg Lys Thr AspGlu Gly Gly Asp Arg Gly Ala Pro 340 345 350 Gly Leu Ala Leu Ala Arg ValArg Ala Pro Ser Asp Thr Thr Asn Gly 355 360 365 Thr Ser Ser Ser Lys GlySer Ser His Asn Lys Gly Gln Arg Ala Ser 370 375 380 Ser Ser Thr Tyr HisArg Gln Arg Arg His Ser Asp Phe Cys Gly Pro 385 390 395 400 Ser Pro AlaPro Leu His Pro Lys Arg Ser Pro Thr Ser Thr Gly Asp 405 410 415 Thr GluLeu Lys Glu Glu Arg Met Pro Gly Arg Lys Ala Ser Cys Ser 420 425 430 AlaVal Gly Ser Gly Ser Arg Gly Leu Pro Pro Ser Ser Pro Met Val 435 440 445Ser Ser Ala His Asn Pro Asn Lys Ala Glu Ile Pro Glu Arg Arg Lys 450 455460 Asp Ser Thr Ser Thr Pro Asn Asn Leu Pro Pro Ser Met Met Thr Arg 465470 475 480 Arg Asn Thr Tyr Val Cys Thr Glu Arg Pro Gly Ser Glu Arg ProSer 485 490 495 Leu Leu Pro Asn Gly Lys Glu Asn Ser Ser Gly Thr Ser ArgVal Pro 500 505 510 Pro Ala Ser Pro Ser Ser His Ser Leu Ala Pro Pro SerGly Glu Arg 515 520 525 Ser Arg Leu Ala Arg Gly Ser Thr Ile Arg Ser ThrPhe His Gly Gly 530 535 540 Gln Val Arg Asp Arg Arg Ala Gly Ser Gly SerGly Gly Gly Val Gln 545 550 555 560 Asn Gly Pro Pro Ala Ser Pro Thr LeuAla His Glu Ala Ala Pro Leu 565 570 575 Pro Ser Gly Arg Pro Arg Pro ThrThr Asn Leu Phe Thr Lys Leu Thr 580 585 590 Ser Lys Leu Thr Arg Arg ValThr Leu Asp Pro Ser Lys Arg Gln Asn 595 600 605 Ser Asn Arg Cys Val SerGly Ala Ser Leu Pro Gln Gly Ser Lys Ile 610 615 620 Arg Ser Gln Thr AsnLeu Arg Glu Ser Gly Asp Leu Arg Ser Gln Val 625 630 635 640 Ala Ile TyrLeu Gly Ile Lys Arg Lys Pro Pro Pro Gly Cys Ser Asp 645 650 655 Ser ProGly Val 660 7 752 PRT Homo sapiens 7 Met Ser Ser Arg Thr Val Leu Ala ProGly Asn Asp Arg Asn Ser Asp 1 5 10 15 Thr His Gly Thr Leu Gly Ser GlyArg Ser Ser Asp Lys Gly Pro Ser 20 25 30 Trp Ser Ser Arg Ser Leu Gly AlaArg Cys Arg Asn Ser Ile Ala Ser 35 40 45 Cys Pro Glu Glu Gln Pro His ValGly Asn Tyr Arg Leu Leu Arg Thr 50 55 60 Ile Gly Lys Gly Asn Phe Ala LysVal Lys Leu Ala Arg His Ile Leu 65 70 75 80 Thr Gly Arg Glu Val Ala IleLys Ile Ile Asp Lys Thr Gln Leu Asn 85 90 95 Pro Ser Ser Leu Gln Lys LeuPhe Arg Glu Val Arg Ile Met Lys Gly 100 105 110 Leu Asn His Pro Asn IleVal Lys Leu Phe Glu Val Ile Glu Thr Glu 115 120 125 Lys Thr Leu Tyr LeuVal Met Glu Tyr Ala Ser Ala Gly Glu Val Phe 130 135 140 Asp Tyr Leu ValSer His Gly Arg Met Lys Glu Lys Glu Ala Arg Ala 145 150 155 160 Lys PheArg Gln Ile Val Ser Ala Val His Tyr Cys His Gln Lys Asn 165 170 175 IleVal His Arg Asp Leu Lys Ala Glu Asn Leu Leu Leu Asp Ala Glu 180 185 190Ala Asn Ile Lys Ile Ala Asp Phe Gly Phe Ser Asn Glu Phe Thr Leu 195 200205 Gly Ser Lys Leu Asp Thr Phe Cys Gly Ser Pro Pro Tyr Ala Ala Pro 210215 220 Glu Leu Phe Gln Gly Lys Lys Tyr Asp Gly Pro Glu Val Asp Ile Trp225 230 235 240 Ser Leu Gly Val Ile Leu Tyr Thr Leu Val Ser Gly Ser LeuPro Phe 245 250 255 Asp Gly His Asn Leu Lys Glu Leu Arg Glu Arg Val LeuArg Gly Lys 260 265 270 Tyr Arg Val Pro Phe Tyr Met Ser Thr Asp Cys GluSer Ile Leu Arg 275 280 285 Arg Phe Leu Val Leu Asn Pro Ala Lys Arg CysThr Leu Glu Gln Ile 290 295 300 Met Lys Asp Lys Trp Ile Asn Ile Gly TyrGlu Gly Glu Glu Leu Lys 305 310 315 320 Pro Tyr Thr Glu Pro Glu Glu AspPhe Gly Asp Thr Lys Arg Ile Glu 325 330 335 Val Met Val Gly Met Gly TyrThr Arg Glu Glu Ile Lys Glu Ser Leu 340 345 350 Thr Ser Gln Lys Tyr AsnGlu Val Thr Ala Thr Tyr Leu Leu Leu Gly 355 360 365 Arg Lys Thr Glu GluGly Gly Asp Arg Gly Ala Pro Gly Leu Ala Leu 370 375 380 Ala Arg Val ArgAla Pro Ser Asp Thr Thr Asn Gly Thr Ser Ser Ser 385 390 395 400 Lys GlyThr Ser His Ser Lys Gly Gln Arg Ser Ser Ser Ser Thr Tyr 405 410 415 HisArg Gln Arg Arg His Ser Asp Phe Cys Gly Pro Ser Pro Ala Pro 420 425 430Leu His Pro Lys Arg Ser Pro Thr Ser Thr Gly Glu Ala Glu Leu Lys 435 440445 Glu Glu Arg Leu Pro Gly Arg Lys Ala Ser Cys Ser Thr Ala Gly Ser 450455 460 Gly Ser Arg Gly Leu Pro Pro Ser Ser Pro Met Val Ser Ser Ala His465 470 475 480 Asn Pro Asn Lys Ala Glu Ile Pro Glu Arg Arg Lys Asp SerThr Ser 485 490 495 Thr Pro Asn Asn Leu Pro Pro Ser Met Met Thr Arg ArgAsn Thr Tyr 500 505 510 Val Cys Thr Glu Arg Pro Gly Ala Glu Arg Pro SerLeu Leu Pro Asn 515 520 525 Gly Lys Glu Asn Ser Ser Gly Thr Pro Arg ValPro Pro Ala Ser Pro 530 535 540 Ser Ser His Ser Leu Ala Pro Pro Ser GlyGlu Arg Ser Arg Leu Ala 545 550 555 560 Arg Gly Ser Thr Ile Arg Ser ThrPhe His Gly Gly Gln Val Arg Asp 565 570 575 Arg Arg Ala Gly Gly Gly GlyGly Gly Gly Val Gln Asn Gly Pro Pro 580 585 590 Ala Ser Pro Thr Leu AlaHis Glu Ala Ala Pro Leu Pro Ala Gly Arg 595 600 605 Pro Arg Pro Thr ThrAsn Leu Phe Thr Lys Leu Thr Ser Lys Leu Thr 610 615 620 Arg Arg Val AlaAsp Glu Pro Glu Arg Ile Gly Gly Pro Glu Val Thr 625 630 635 640 Ser CysHis Leu Pro Trp Asp Gln Thr Glu Thr Ala Pro Arg Leu Leu 645 650 655 ArgPhe Pro Trp Ser Val Lys Leu Thr Ser Ser Arg Pro Pro Glu Ala 660 665 670Leu Met Ala Ala Leu Arg Gln Ala Thr Ala Ala Ala Arg Cys Arg Cys 675 680685 Arg Gln Pro Gln Pro Phe Leu Leu Ala Cys Leu His Gly Gly Ala Gly 690695 700 Gly Pro Glu Pro Leu Ser His Phe Glu Val Glu Val Cys Gln Leu Pro705 710 715 720 Arg Pro Gly Leu Arg Gly Val Leu Phe Arg Arg Val Ala GlyThr Ala 725 730 735 Leu Ala Phe Arg Thr Leu Val Thr Arg Ile Ser Asn AspLeu Glu Leu 740 745 750 8 688 PRT Homo sapiens 8 Met Ser Ser Arg Thr ValLeu Ala Pro Gly Asn Asp Arg Asn Ser Asp 1 5 10 15 Thr His Gly Thr LeuGly Ser Gly Arg Ser Ser Asp Lys Gly Pro Ser 20 25 30 Trp Ser Ser Arg SerLeu Gly Ala Arg Cys Arg Asn Ser Ile Ala Ser 35 40 45 Cys Pro Glu Glu GlnPro His Val Gly Asn Tyr Arg Leu Leu Arg Thr 50 55 60 Ile Gly Lys Gly AsnPhe Ala Lys Val Lys Leu Ala Arg His Ile Leu 65 70 75 80 Thr Gly Arg GluVal Ala Ile Lys Ile Ile Asp Lys Thr Gln Leu Asn 85 90 95 Pro Ser Ser LeuGln Lys Leu Phe Arg Glu Val Arg Ile Met Lys Gly 100 105 110 Leu Asn HisPro Asn Ile Val Lys Leu Phe Glu Val Ile Glu Thr Glu 115 120 125 Lys ThrLeu Tyr Leu Val Met Glu Tyr Ala Ser Ala Gly Glu Val Phe 130 135 140 AspTyr Leu Val Ser His Gly Arg Met Lys Glu Lys Glu Ala Arg Ala 145 150 155160 Lys Phe Arg Gln Ile Val Ser Ala Val His Tyr Cys His Gln Lys Asn 165170 175 Ile Val His Arg Asp Leu Lys Ala Glu Asn Leu Leu Leu Asp Ala Glu180 185 190 Ala Asn Ile Lys Ile Ala Asp Phe Gly Phe Ser Asn Glu Phe ThrLeu 195 200 205 Gly Ser Lys Leu Asp Thr Phe Cys Gly Ser Pro Pro Tyr AlaAla Pro 210 215 220 Glu Leu Phe Gln Gly Lys Lys Tyr Asp Gly Pro Glu ValAsp Ile Trp 225 230 235 240 Ser Leu Gly Val Ile Leu Tyr Thr Leu Val SerGly Ser Leu Pro Phe 245 250 255 Asp Gly His Asn Leu Lys Glu Leu Arg GluArg Val Leu Arg Gly Lys 260 265 270 Tyr Arg Val Pro Phe Tyr Met Ser ThrAsp Cys Glu Ser Ile Leu Arg 275 280 285 Arg Phe Leu Val Leu Asn Pro AlaLys Arg Cys Thr Leu Glu Gln Ile 290 295 300 Met Lys Asp Lys Trp Ile AsnIle Gly Tyr Glu Gly Glu Glu Leu Lys 305 310 315 320 Pro Tyr Thr Glu ProGlu Glu Asp Phe Gly Asp Thr Lys Arg Ile Glu 325 330 335 Val Met Val GlyMet Gly Tyr Thr Arg Glu Glu Ile Lys Glu Ser Leu 340 345 350 Thr Ser GlnLys Tyr Asn Glu Val Thr Ala Thr Tyr Leu Leu Leu Gly 355 360 365 Arg LysThr Glu Glu Gly Gly Asp Arg Gly Ala Pro Gly Leu Ala Leu 370 375 380 AlaArg Val Arg Ala Pro Ser Asp Thr Thr Asn Gly Thr Ser Ser Ser 385 390 395400 Lys Gly Thr Ser His Ser Lys Gly Gln Arg Ser Ser Ser Ser Thr Tyr 405410 415 His Arg Gln Arg Arg His Ser Asp Phe Cys Gly Pro Ser Pro Ala Pro420 425 430 Leu His Pro Lys Arg Ser Pro Thr Ser Thr Gly Glu Ala Glu LeuLys 435 440 445 Glu Glu Arg Leu Pro Gly Arg Lys Ala Ser Cys Ser Thr AlaGly Ser 450 455 460 Gly Ser Arg Gly Leu Pro Pro Ser Ser Pro Met Val SerSer Ala His 465 470 475 480 Asn Pro Asn Lys Ala Glu Ile Pro Glu Arg ArgLys Asp Ser Thr Ser 485 490 495 Thr Pro Asn Asn Leu Pro Pro Ser Met MetThr Arg Arg Asn Thr Tyr 500 505 510 Val Cys Thr Glu Arg Pro Gly Ala GluArg Pro Ser Leu Leu Pro Asn 515 520 525 Gly Lys Glu Asn Ser Ser Gly ThrPro Arg Val Pro Pro Ala Ser Pro 530 535 540 Ser Ser His Ser Leu Ala ProPro Ser Gly Glu Arg Ser Arg Leu Ala 545 550 555 560 Arg Gly Ser Thr IleArg Ser Thr Phe His Gly Gly Gln Val Arg Asp 565 570 575 Arg Arg Ala GlyGly Gly Gly Gly Gly Gly Val Gln Asn Gly Pro Pro 580 585 590 Ala Ser ProThr Leu Ala His Glu Ala Ala Pro Leu Pro Ala Gly Arg 595 600 605 Pro ArgPro Thr Thr Asn Leu Phe Thr Lys Leu Thr Ser Lys Leu Thr 610 615 620 ArgArg Val Thr Leu Asp Pro Ser Lys Arg Gln Asn Ser Asn Arg Cys 625 630 635640 Val Ser Gly Ala Ser Leu Pro Gln Gly Ser Lys Ile Arg Ser Gln Thr 645650 655 Asn Leu Arg Glu Ser Gly Asp Leu Arg Ser Gln Val Ala Ile Tyr Leu660 665 670 Gly Ile Lys Arg Lys Pro Pro Pro Gly Cys Ser Asp Ser Pro GlyVal 675 680 685

1. Nucleic acid that codes for a neuronal serine threonine proteinkinase, selected from a) a nucleic acid, with one of the sequencesaccording to SEQ ID NOs. 1-4 and a nucleic acid that codes for a proteinwith a sequence according to one of SEQ ID NOs. 5-8; b) a nucleic acidthat hybridised with a nucleic acid according to a); c) a nucleic acidwhich, taking account of the degeneration of the genetic code, wouldhybridise with a nucleic acid according to a); d) derivatives of anucleic acid according to a)-c) obtained by substitution, addition,inversion and/or deletion of one or more bases, and e) a nucleic acidcomplementary to a nucleic acid according to a)-d).
 2. Nucleic acidaccording to claim 1, characterised in that it codes for a protein thatexhibits at least 60% identity to one of the sequences with SEQ ID NOs.5-8.
 3. Nucleic acid according to one of claims 1 or 2, characterised inthat it is at least 60% identical to the coding sections of the sequenceaccording to one of SEQ ID NOs. 1-4.
 4. Nucleic acid according to one ofclaims 1-3, characterised in that it codes for a protein sequenceaccording to one of SEQ ID NOs. 5-8.
 5. Nucleic acid according to one ofclaims 1-4, characterised in that it is a DNA.
 6. Fragment of a nucleicacid according to one of claims 1-5, characterised in that, in antisenseorientation to a promoter, it can inhibit the expression of a serinethreonine protein kinase in a host cell.
 7. Fragment according to claim6, characterised in that it comprises at least 10 nucleotides,preferably at least 50 nucleotides, particularly preferably at least 200nucleotides.
 8. Construct containing a nucleic acid according to one ofclaims 1-5 and/or a fragment according to one of claims 6 or 7,preferably under the control of the expression-regulating elements. 9.Construct according to claim 8, characterised in that the nucleic acidor the fragment is in antisense orientation to the control element. 10.Construct according to one of the foregoing claims, characterised inthat it is present in a plasmid.
 11. Host cell containing a nucleic acidaccording to one of claims 1-5 and/or a fragment according to one ofclaims 6 or 7 and/or a construct according to one of claims 8-9. 12.Host cell according to claim 11, characterised in that it is selectedfrom bacteria, yeast cells and mammalian cells.
 13. Mammalian tissue,organ and/or transgenic mammal containing a nucleic acid according toone of claims 1-5 and/or a fragment according to one of claims 6 or 7and/or construct according to one of claims 8-10.
 14. Host cell and/ormammalian tissue and/or mammalian organ and/or transgenic mammalaccording to one of the foregoing claims, characterised in that thenucleic acid or fragment or construct is integrated in a position of thegenome that does not correspond to its natural position.
 15. Proteinobtainable by expression of a nucleic acid according to one of claims1-4 in an expression system, preferably a host cell.
 16. Proteinaccording to claim 15, characterised in that it is selected from aprotein with one of the sequences according to SEQ ID NOs. 5-8 andproteins that are at least 60% identical to one of the said proteins.17. Antibody that reacts with a protein according to one of claims 15 or16.
 18. Inhibitor of the protein kinase activity of the proteinaccording to one of claims 15 or
 16. 19. Phosphorylation substrate ofthe protein kinase activity of the protein according to one of claims 15or
 16. 20. Intracellular protein interaction partner of the proteinaccording to one of claims 15-16, obtainable with the aid of theYeast-Two-Hybrid selection system, or immunoprecipitations. 21.Diagnostic kit containing a nucleic acid according to one of claims 1-5and/or a fragment according to one of claims 6 or 7 and/or a constructaccording to one of claims 8-10 and/or a protein according to one ofclaims 15 and 16 and/or an antibody according to claim
 17. 22. Methodfor the diagnosis of a risk of apoplexy, comprising the stepdetermination of the level of expression of a nucleic acid according toone of claims 1-5 or of the quantity of protein according to one ofclaims 15 or 16 in a patient sample.
 23. Method according to claim 22,characterised in that the patient sample is brought into contact with anucleic acid according to one of claims 1-5 and/or an antibody accordingto claim
 17. 24. Method for the identification and/or isolation of aprotein kinase inhibitor, characterised in that the protein kinaseactivity of a protein according to one of claims 15 or 16 is determinedin the presence and absence of the test substance and the substance thusidentified as suitable is isolated.
 25. Protein kinase assay,characterised in that the protein kinase investigated comprises aprotein according to one of claims 15 or
 16. 26. Pharmaceuticalcomposition containing a nucleic acid, a fragment, a construct, a hostcell, a protein, an antibody and/or an inhibitor according to one ofclaims 1-18.
 27. Use of a nucleic acid, a fragment of a nucleic acid, aconstruct, a host cell, a protein, an antibody and/or an inhibitoraccording to one of claims 1-18 for the prophylaxis and/or therapy ofneurological diseases, including stroke, multiple sclerosis, Parkinson'sdisease, amyotrophic lateral sclerosis, heterodegenerative ataxias,Huntington's disease,. neuropathies and epilepsies and/or tumourdiseases, particularly carcinomas and/or immunological diseases,including autoimmune diseases, atopies and/or HIV infections and/orinfections by other immunotropic viruses and/or acute and/or chroniclymphatic leukaemia, and/or acute and/or chronic myeloid leukaemiaand/or primary chronic polyarthritis and/or Crohn's disease and/orColitis ulcerosa.
 28. Use of a nucleic acid, a fragment, a construct, ahost cell, a protein, and/or an antibody according to one of claims 1-17for the identification of protein kinase inhibitors.
 29. Use of aninhibitor of the protein kinase activity of a protein according to oneof claims 15 or 16 for the prophylaxis and/or therapy of a neurologicaldisease and/or a neoplastic disease.
 30. Use of a nucleic acid and/or aprotein according to one of the foregoing claims as a point of attackfor a pharmaceutical active substance.